Basic Processes of Molecular Biology

Basic Processes of Molecular Biology

Core Course # MM 702

Dr. Sonia Siddiqui

Dr. Panjwani Centre For Molecular Medicine and Drug Research (PCMD)

Meselson-Stahl experiment

DNA replication is Semiconservative

Bidirectional DNA replication begins at an Origin

DNA Replication and Recombination

Synthesis of DNA molecule: in 3 steps

1- Initiation

2- Elongation

3-Termination

These processes required many different types of enzymes

1- DNA replicase system or replisome

2- Helicases

3- Topoisomerases

4- Primase

5- DNA ligases

Initiation

E.Coli DNA replication origin called OriC containing 246 bp.

For replication specific sequencing are present which is recognized by the enzymes involved in the initiation:

1- 9 bp sequences on which DnaA protein binds

• DnaA-binding sites (I sites), IHF (Integration host factor) and FIS (factor for inversion stimulation).

2- 13 bp rich A=T sequences on which DNA unwinding element (DUE)

IHF FIS

Initiation

• DnaA protein is a member of the AAA+ ATPase protein family

Function: formation of oligomers and hydrolyze ATP (do things slowly)

• 8 DnaA protein molecules in ATP bound form makes a helical complex emcompassing the R and I sites in oriC.

• It has high affinity towards R sites than I

• It binds to R sites in ATP or ADP-bound form whereas It binds to I sites in only ATP-bound form

Initiation

How DnaC docks DnaB protein?

• Hexamer of each DnaC subunit bound with ATP binds with hexameric ring-shaped DnaB helicase.

• This interaction of DnaB-DnaC opens DnaB ring, further interaction required DnaA

• 2 out of 6 hexamer of DnaB are loaded on DUE on to each strand.

• DnaC+ATP hydrolyzed, releasing DnaC and DnaB bound to the DNA.

Key step in replication: DnaB helicase docking on the DNA

DnaB unwinds the DNA from 5’→ 3’ of single stranded DNA, both strands moves in opposite direction.

• This DNA with DnaB helicase has two replication forks

• DNA polymerase III holoenzyme is linked via epsilon subunits

• Many other single stranded DNA-binding protein (SSB) are involved that binds on each of the DNA strand at the fork

• Simultaneously DNA gyrase or DNA topoisomerase II relieves the tension in the DNA molecule at the fork

The oriC DNA is methylated by Dam methylase at N6 of adenine 5’ GATC region (palindromic sequence)

• Completion of DNA replication the oriC region of DNA is methylated but the newly strand is not

• The hemimethylated oriC sequences are now ready to interact with the plasma membrane with the help of a protein called SeqA

• OriC is released from the plasma membrane and SeqA is dissociates and DNA is fully methylated by Dam methylase

Elongation of the DNA: Leading and Lagging strand synthesis

Leading strand synthesis:

• It begins with the synthesis by primase RNA primer (DnaG,10-60 nucleotide) at the fork

• DnaG + DnaB helicase, primer synthesis takes place opposite in the direction of helicase movement

• DnaB helicase moves along the DNA strand, the lagging strand

• dNTs keep adding to the DNA strand by DNA polymerases III + DnaB complex moving on the opposite strand

Lagging strand: Okazaki fragments

• On the other hand, Lagging strand synthesis starts by the formation of okazaki fragments replication direction is always from 5’- 3’

• Primase synthesize RNA primer and DNA polymerase III + DnaB adds dNTs to the lagging strand like in leading strand

Clamp loading complex of DNA polymerase III

• It contains two subunits along with the subunits along with the ….. Subunits + AAA + ATPase

• This whole complex binds to ATP and the new β sliding clamp

• This creates a stretch on the dimeric clamp, opening up the ring at one subunit interface

• Lagging strand slipped into the ring via breaking

• Clamp loader hydrolyzes ATP, releasing the β sliding clamp

Okazaki fragments:

Okazaki fragments synthesis complexity:

• DNA polymerase III forms a dimer around both the strands bringing the strand close together

• DnaB + DnaG complex forms at the replication fork called Replisome

• DNA polymerase III has two sets of core subunits one synthesize the leading strand while the other synthesize the Okazaki fragments on the lagging strand

• It is noted that at the Primosome there is β sliding clamp complex is present which is prepared by DNA polymerase III

DNA ligation by Ligases

DNA Ligases

• Ligase enzyme catalyzes the formation of a phosphodiester bond between a 3’ hydroxyl at the end of one DNA strand and a 5’ phosphate at the end of another strand

• Via adenylation the phosphate can be activated

Properties of DNA ligase

• It is isolated from viruses and eukaryotes use ATP however, DNA ligases from bacteria are different

a) Many DNA ligase use NAD+ a cofactor that normally functions in hydride transfer reactions, a source of the AMP activating group

b) DNA ligase can also be very useful in DNA recombination experiments

Replication in Eukaryotes

Cell –Cycle control System and Activated Protein Kinases

• Cyclin dependent kinases (cdks) a protein kinases which actually regulates major events of cell cycle such as DNA replication, mitosis and cytokinesis

• In crease cdks levels during and at the beginning of mitosis leads to the increase phosphorylation of proteins that controls chromosome condensation, nuclear envelop breakdown and spindle assembly

• However cdks activity is control by many complexes and proteins such as cyclins, cyclin activating kinases (CAK), cdk inhibitor protein (CKI), SCF, and Anaphase-promoting complex (APC), cdk25 and wee1

Cyclins-cdks complex

• cdks require cyclins for their activation

• Cyclins are synthesized and degraded in each cell cycle

• cdks level remain normal through out the cell cycle, however changes in the levels of cyclins causes the assembly of cyclin-cdk complexes- leads to the activation and triggering of the cell-cycle events

• There are four classes of cyclins G1/S-cyclins, S-cyclins, M-cyclins and G1 cyclins

• Mode of activation is each complex phosphorylate the target substrate proteins and can change the activity of activation according the levels of substrate that changes during or after the cell cycle

• CAK activates the cyclin-cdk complex by phosphorylating an a.a near the cdk active site—which eventually activates the target protein and induce sp cell-cycle activity

Regulation of cyclin-cdk complex

• The activity of the complex can be inhibited by phosphorylation via Wee1, a protein kinase and activation can be done by a phosphatases which dephosphorylate the complex via cdc25

• The activity of the complex can be regulated by another kinases cdk inhibitor proteins (CKIs), which controls mainly S and G 1 phases . Upon binding conformational changes takes place and makes it inactive

Cyclical proteolysis and cell-cycle control system

• The rate limiting step in cyclin destruction is the final ubiquitin-transfer

reaction performed by 2 ubiquitin ligases, APC complex and SCF

• SCF in S and G1 phase ubiquitinate the complex G1/S-cyclins and

certain CKI that are involve in S phase initiation

• However M phase is controlled by APC complex, it proteolyzed and

ubiquitinites cyclins and other proteins involve in M phase

• SCF activity is constant throughout the cell cycle, however APC levels

changes with cell-cycle stages

Cell –Cycle control and Transcriptional Regulation

• In more complex cell cycle, cyclins are controlled not only by there levels but by controlling at the gene transcription level and its synthesis.

Intracellular control of cell cycle events

• The maintenance of each phase of cell-cycle that is G phase fusing with S phase fusing with G1 phase fusing with M phase and then G phase again, requires highly skilled and accuracy and constant adding of activating substrates to maintain the smooth overlap of the phases at different stages of cell-cycle

• For eg cdc6 a regulator protein, its level increases only in G1 phase where it is required to bind with a complex with closely related proteins, minichromosomal maintenance proteins (Mcm) , resulting in the formation of a large pre-replicative complex or pre-RC complex

Intracellular control of cell cycle events

• The activation of the S-cdk in late G1 initiates DNA replication, another kinases phosphorylate the Pre-Rc complex

• S-Cdk helps cdc6 protein to dissociate from ORC after an origin is fired--- this leads to the disassembly of pre-RC which prevents replication from occurring again at the same origin

• Secondly It prevents cdc6 and Mcm proteins from reassembling at any origin

• It phosphorylates the cdc6, and triggers the ubiquitinylation by the SCF protein

• S-Cdk also phoshorylates certain Mcm proteins which triggers their export from the nulceus, further proving that Mcm complex cannot bind to the replication origin

• At the end all Cdk levels becomes zero, this dephosphorylate the cdc6 and Mcm proteins allow pre-Rc complex assembly to occur once again

Replication in Eukaryotes Cells

Cyclin dependent kinases (CDKs) regulation control over DNA replication

• The cyclins destruction by Ubiquiton-dependent proteolysis at the end of M phase

• In the absence of CDKs the pre-replicative complexes (pre-RCs) can be formed on replication sites

• In fast growing cells, this pre-RCs complex forms at the end M phase. Pre-RCs are called licensing

• In eukaryotes the replication started by the formation of a mini chromosome maintenance (MCM) proteins

• Many diff. types of MCM proteins exits like MCM2-MCM7 helicase also resembles like DnaB helicase, loads on ORC along CDC6 (cell division cycle) and CDT1 (Cell division transcript 1)

• Replication requires the S phase, cyclin-cyclin dependent kinase complexes and CDC7-DBF4

• For replication both complexes must be together and the phosphorylating proteins on the pre-RCs complex

Control of replication achieved by inhibiting the synthesis of more complexes by CDK2 and other cyclins

Termination

• Ter sequence trap the replication fork

• Ter is for protein Tus (terminus utilization substance) binding

• Ter-Tus complex works per replication cycle upon collision of either fork

• Ter prevent over replication by replication fork and halts upon collision of other fork

• The sequences that comes in between Ter-Tus will be replicated only , making catenane circular chromosomes

Mechanism of DNA Repair: Mismatch Repair

Early steps of methyl-directed mismatched repair

• MutL + MutS complex at 5’ GATC binds to all mismatched base pairs

• MutH + MutS binds to GATC

• MutL + MutS complex creates a loop on DNA at both sides

• MutH has specific endonuclease activity cleaves unmethylated GATC seq.

• MutH cleaves only G at 5’ side of GATC seq.

Finishing of methyl-directed mismatched repair

When the mismatching is at 5’

• Ummethylated strand is degraded in 3’-5’

• This requires many enzymes

•1-DNAhelicase II

•2- SSB

•3- Exonuclease I OR X

•4- DNA polymerase III

•5-DNA ligase

When the mismatching is at 3’

• Exonuclease will be either VII (for degradation in to 3’-5’ or 5’-3’) OR RecJ nuclease (degrades sDNA in 5’-3’)

Mechanism of DNA Repair: Base Excision Repair

• DNA glycosylases recognize the AP and abasic sites (generated by the cleavage of adenine and cytosine deamination)

• Uracil DNA glycosylase removes uracil only from DNA

• Enzyme recognize thymidine base from Uracil in DNA ie why DNA has thymidine and not uracil

Base-excision repair pathway

• Humans have 4 types of DNA glycosylase with different specificities

• Humans also has hSMUG1 which also removes U

• TDG and MBD4 removes U or T present with G

• Other DNA glycosylase recognize and removes formamidoprymidine and 8-hydroxyguanine (arised from purine deamination)

• It also removes hypoxanthine and alkylated bases like 3-methyladenine and 7-methylguanine

Mechanism of DNA Repair: Nucleotide-Excision Repair

6th 22nd 8th 5th

Excinucleases DNA repair in E. Coli

• Enzymatic complex ABC excinuclease (can create two cleavages)

•Subunits:

1- UvrA Mr 104,000

2- UvrB Mr 78,000

3- UvrC Mr 68,000

• Repair mechanism like nucleotide-excision repair and base-excision repair is tied to transcription in eukaryotes

• This pathway helps to repair DNA from various carcinogens like benzo[ά] pyrene-guanine, cyclobutane pyrimidine dimers and 6-4 photoproducts

Eukaryotic excinucleases DNA repair system : DNA damages caused by cigarette smoke can be repair by this repair mechanism

Mechanism of DNA Repair: Direct Repair

Direct Repair: Pyrimidine dimers by photolyases

• O6-methylguanine forms in the presence of alkylating agents

• This makes pairs with thymine instead of cytosine leading mismatched A-T and C-G bonds

• Repairment is achieved by O6-methylguanine-DNAmethyltransferase

• This enzyme transfer a methyl group of O6-methylguanine to one of its own Cys residues

Direct Repair: Damage caused by alkylating agents on nucleotide

Direct Repair: Damage caused by alkylating agents on nucleotide

Direct repair: Alkylated bases by AlkB

• 1-methyladenine and 3-methylcytosine is repaired by ά-ketoglutarate-Fe2+ -dependent dioxygenase superfamily

• In this repair A and C residues which sometimes becomes methylated in ssDNA, which affects correct base pairing

• In E. coli, oxidation demethylation of these bases is mediated by AlkB protein, a member of this enzyme superfamily

Consequences of Replication fork + DNA damage

• Lesion in dsDNA and ssDNA appears when the damaged DNA didn’t find complementary strand for the correct synthesis or when a replication fork encounters unrepaired DNA lesion

Error-prone translesion DNA synthesis:

• The DNA repair under this pathway is less accurate

with high mutation

• In bacteria this pathway is ON only when there is a

•continuous damage to the cell’s DNA (oxidation or stress)

like SOS response

• The production of normally present proteins UvrA and UvrB

Increases

• Other proteins UmuC and UmuD activated

• UmuD protein regulated by SOS response and cleaved

in to UmuD’

• UmuD’+ UmuC complex to form a specialized DNA polymerase V, helps in replication

• Still difficult to make base pairing, hence can have many chances of error

Genes Induced as part of the SOS response in E.coli


• Desperate strategy from a cell to start the synthesis of UmuC and UmuD initiated by a SOS response resulting in the activation of DNA polymerase V is a deliterious. Many daughter cell dies due to the activation of this type of repair mechanism

• Continuous degradation of the DNA molecule also activates RecA protein that binds ssDNA on one chromosomal location and binds with DNA polymerase V at distant sites.

• DNA polymerase η (eta) found in all eukaryotes and initiates TLS primary β, iota and λ have specialized role in base- excision repair

• These enzymes also have 5’-deoxyribose PO4 lyase activity

• After the removal of base by glycosylase and PO4 group by AP endonuclease, Polymerase removes the abasic site (5’ PO4) and fill in the short gap

• This leads to the reduction in DNA polymerase η activity due to the short length of DNA


You tube Links- DNA Repair

http://www.youtube.com/watch?v=kp0esidDr-c&feature=related

http://www.youtube.com/watch?v=nPS2jBq1k48&feature=related

http://www.youtube.com/watch?v=nPS2jBq1k48&feature=related

http://www.youtube.com/watch?v=y16w-CGAa0Y&feature=related

http://www.youtube.com/watch?v=y16w-CGAa0Y&feature=related

http://www.youtube.com/watch?v=nUzyrBC0tTY

http://www.youtube.com/watch?v=idbGJsDXDFo&NR=1

http://www.youtube.com/watch?v=kp0esidDr-c&feature=related

DNA Recombination

• Homologous Genetic Recombination: Involves genetic exchange between two molecules DNA having similar sequences

• Site-specific recombination: Exchange occurs only at particular sequence on a DNA

• DNA transposition: Short segment of DNA in which chromosome moves from one location to another

Homologous Genetic Recombination: Base-pairing between two homologous DNA molecule

• Meiosis characteristics

• Two Different chromosome from two homologous DNA cross over= DNA break and ends join to their opposite partners to re-form two intact helices

Both of these helices contains half and half part of both the DNA.

• The site of cross over or the exchange of the part of DNA molecules can occur anywhere in the entire DNA having homologous nt sequences in both DNA molecules

Homologous Genetic Recombination: Base-pairing between two homologous DNA molecule

• This type of recombination occurs when a long region of nt sequences on both the strands are in a match

• The point at which the cross over occur is called DNA synapsis

Qs arises that how both the strands recognize the site to start cross over ???

Homologous Genetic Recombination: Meiotic Recombination by dsDNA breaks

• The break in PO4 diester bond attracts the other DNA helix to form base pairing thus forms a synapsis

• It is thought that these strands search base pairing on another DNA strand having matching or homologous sequences

• Leading to the formation of a point or joint between maternal and paternal chromosome

Homologous Genetic Recombination: Meiotic Recombination by dsDNA breaks

Qs How the synthesis of ds homologous DNA molecule starts to begin the DNA synapsis

Homologous Genetic Recombination: DNA hybridization reactions model

• When a double helix DNA re-forms from a ssDNA. This is also called DNA renaturation or hybridization

• This step follows a quickly zipping up the DNA molecule base pairing to the maximum

• Annealing is required bc the DNA is in unfolded form

• Some times ssDNA strand folds back on itself for the base pairing like a short hairpin

• This is critical condition for the cells i.e. why Single-Strand binding protein is required

Homologous Genetic Recombination: RecA and its Homologs

• RecA has multiple DNA binding sites and catalyzes multistep synapsin formation

• Before this the homology between ssDNA and the region in dsDNA strand is identified by making transient base pairing

• Once synapsis starts, short heteroduplex regions have begun to make base pairing to the longer distances via process called branch migration

• This branch point can occur at any point where two single DNA strands with the same sequences are attempting to pair with the same complementary strand

• RecA is DNA-dependent ATPase, with ATP hyrolyzing site

• RecA tightly binds with DNA+ATP rather than DNA+ADP

Homologous Genetic Recombination: RecA and its Homologs

• ATP continuously added to one end of the RecA protein filaments, while ATP hydrolyzes to ADP

• Therefore DNA share some dynamics of cytoskeleton filaments actin or tubulin

Homologous Genetic Recombination: Holliday Junction

• Holiday junction contains two of four dsDNA strand that are crossing and forming the base pairs

• An Holliday junction produces an open, symmetrical structure

• Further isomerization can interconvert the crossing and non crossing strands, producing a structure that is otherwise the same when it starts

• The formation of holliday junction requires ATP hydrolysis used by sets of proteins

Homologous Genetic Recombination: Gene Conversion

Gene Conversion:

• DNA sequence information is transferred from one DNA helix to another DNA helix whose sequence is now altered due to the transfer

• It could happen by a homologous recombination process that juxtaposes two homologous dsDNA helices OR

• Short piece of DNA synthesis occur for the new allele base pairing

Homologous Genetic Recombination: Gene Conversion

• Simply a heteroduplex joint forms in which both DNA helices have different nt sequences and are not matched

• Unmatched sequences are removed by DNA repair mechanism, resulting in the formation of an extra copy of DNA sequence on the opposite strand

• Then the same gene conversion process occur without crossover

Homologous Genetic Recombination: Outcomes in Meiosis and Mitosis

• Either outcome in general recombination

1- The DNA synthesis involved convert some of the genetic information at the site of the double stranded break to that of the homologous chromosome

2- If these regions represent different alleles of the same gene

3- Then the nucleotide sequence in the broken helix is converted to that of unbroken helix, resulting a gene conversion

Homologous Genetic Recombination: Prevention of Promiscuous Recombination b/w two poorly matched DNA sequences

• Mismatch proofreading system normally recognizes the mispaired bases in an initial strand exchange

• These type of mechanism protect bacteria or cells from invading foreign DNA to form base pairs with the host DNA

Site-Specific Recombination

• It can alter gene order and also add new information to the genome

• In this recombination the genes position changes within one chromosomes OR to another chromosomes

• In this specialized nt sequences moves on a nonhomologous sites within a genome. These movable sequences are called mobile genetic elements

• These elements differ in size and ranges from 100s or 10,000s nt base pairs

• These elements are present in all cells from E coli. to humans

Site-Specific Recombination

• Importance of this type of recombination is to produce many genetic variants on which evolution depends

Reason:

These movable variants can also alter the adjacent host cells genome DNA sequences, by carry them to another site

Site-Specific Recombination: Transpositional or Conservative movement

• Tranpositional Recombination :

1- Site-specific recombination requires enzymes and specific DNA sites

2- Does not involve the formation of heteroduplex DNA

3- It involves the ends of the broken DNA segments in chromosome + these ends should be attached at one of many different nonhomologous target DNA sites

• Conservative Site-Specific Recombination

1- This involves the formation of a short heteroduplex joint

2- Due to this it requires short same DNA sequences in donor and recipient DNA molecule

DNA Transposones: Capable of injecting mobile genetic elements in to any DNA sequences

• Transposons, are moveable genetic elements capable of injecting themselves in to many DNA sites

• Transposase enzyme is encoded by transposon

Mechanism of action: This enzyme first loosen the transposons from the DNA and then insert it in to the new DNA sites. Homology b/w the site and the end of the DNA is not an issue

• Usually they move very rarely and this is why they are difficult to detect

• They can be divided in to three classes

1- DNA-only transposons

2- Retroviral-like retrotransposons

3- Nonretroviral retrotransposons

DNA Transposons

Functions and specificities of DNA Transposons

1- DNA-only transposons

In this the mobile element DNA is cut out from the donor DNA and joined in the target DNA by transposase. Hence exists as a DNA all life

2- Retroviral-like retrotransposons

They don’t move directly rather they need RNA polymerase to transcribe the mobile element sequence into RNA. Then RNA transcriptase synthesize DNA from this RNA using it as a template. Then this DNA will incorporate in to new DNA sites of the target DNA, by an enzyme integrase.

3- Nonretroviral retrotransposons

It also requires RNA transcriptase to convert RNA in to DNA. But here RNA is directly involved in the transposition reaction

DNA-only Transposons

DNA-only Transposons

Transpositional Site-specific Recombinant: Viruses mode of incorporation in to the host DNA

Retroviral-like Retrotransposons Resemble Retroviruses, but Lack a Protein Coat

Nonretroviral Retrotransposons

• Most of the human DNA is composed of LINE element (Long interspersed nuclear element)

• Although most of the copies of L1 element are immobile, a few retain the ability to move

• Some times movement of these elements causes a disease for eg in Hemophilia, L1 insertion in to the gene encoding blood clotting factor VIII

• Nonretroviral retrotransposons can also be found in Yeast mitochondria, mammals and insects

• They move via the help of endonuclease complex and reverse transcriptase

• Other DNA repeats that lacks either endonucleases or reverse transcriptase in their nt sequences uses cell’s endonucleases and reverse transciptase, including L1 elements

• For eg Alu elements lacks endonucleases or reverse transcriptase genes, still it has amplified and becomes the major part of the human genome

• Alu and L1 genes sequences are closely related to the mouse sequences, but their incorporation in mouse sequences is different than in humans

Reversible rearrangement of DNA = Conservative Site-specific recombination

Conservative Site-specific recombination: An example

• Bacteriophage lambda virus infects a bacterial DNA and synthesize an encoded enzyme integrase

• Viral DNA covalently joins with the bacterial (host) chromosome, and becomes a part of host DNA and replicates

• Integrase enzyme act so by recognizing special site on the host as well as on the viral DNA for the joining of the two strand

• To reverse this link between two strands the same mechanism can be use to excise the DNA

• By getting specific signals from the cells lambda virus DNA jumps and leaves the sites on chromosome and multiply rapidly in the bacteria

• This excision is catalyze by excisionase

Conservative Site-specific recombination: An example

The life cycle of bacteriophage Lambda

Conservative Site-Specific Recombination Can be Used to Turn Genes On or Off

RNA Transcription

• RNA polymerase binds to the bacterial DNA in spe region known as promoter

• The polymerase, using its σ factor recognizes this DNA seq by making specific

contacts with the portions of the bases that are exposed on the outside of the helix

• After RNA polymerase binds tightly to the promoter DNA in this way, it opens up

the double helix to expose short stretches of nt on each strand

• Instead of DNA helicase, here the nick does not require the hydrolysis of ATP,

both DNA and polymerase structurally changes themselves in a more energetically

favor state.

• RNA polymerase I transcribed 5.8 S, 18S, and 28S rRNA genes

• RNA polymerase II transcribed all protein-coding genes,plus snoRNA

genes and some snRNA genes

• RNA polymerase III transcribed tRNA genes, 5S rRNA genes, some

snRNA genes and genes for other small RNAs

RNA polymerases in Eukaryotic cells

Transcription In Eukaryotes: RNA polymerase II

Initiation of the RNA transcription by RNA polymerase II

• All the transcription factors assembled on the

promoter region which is recognized by the RNA

ployII

• For transcription initiation complex, TFIID

creates a big loop in the DNA strand so that all

the factors can join and assembled at the

promoter region for the protein assembly steps.

• TFIIH has a kinase activity as well as a helicase

property

• The termination of RNA is carried out TFIIH

which adds phosphate groups to the tail of the

RNA polymerase known as CTD or C-terminal

domain

RNA processing (Post transcriptional modification)

RNA Capping

• The 5’ end of the new RNA molecule is modified by the addition of a cap contains a modified Guanine nucleotide

• The capping reaction is catalyzed by

1- Phosphatase which removes a PO4 from 5’ end of the RNA

2- a guanyl transferase that adds GMP in a reversal linkage 5’-5’ instead to 5’-3’

3- Methyl transferase that adds a methyl group to the guanosine

mRNA molecule

5’ Capping of the mRNA

RNA factory

Removal of Introns from the newly synthesized Pre- mRNA

• Eukaryotes genes were found in many coding sequence known as expressed or axon and intervening sequences or introns

• However both are transcribed in to RNA

• In the two sequential phosphoryltransfer reaction or transesterfications join the exons while removing the introns as a “lariat”

RNA Splicing Maschinery

• It consists of 5 additional RNA mols. + > 50 proteins + requires many ATP mols/ splicing

• This event should be highly accurate, any mistake in splicing could harm or kill the cells

• Importance of introns= To produce new types of proteins + helps in genetic recombination to combine the axons of different genes + with the same genes

• Introns can be 10 nt to 100,000 nt long

• Picking the correct place for their removal is not easy

• This is done at 3 positions

1- 5’ splice site

2- 3’ splice site

3- Branch point at the middle of the intron (excised lariat)

• These all positions have similar consensus nt sequences which mark splicing positions

• Still difficult to remove all

Splicing positions on Introns

Spliceosome

• RNA molecules are involved instead of proteins for splicing

• Short RNA mols (200 nt) and named U1, U2, U4, U5, U6

• They all form a complex called as snRNAs (small nuclear RNAs)

• snRNAs work with 7 protein subunits, snRNP (small nuclear ribonulceo protein)

• snRNAs + snRNPs forms the core of spliceosome

mRNA splicing mechanism

RNA-RNA rearrangements

Proper splice sites in Pre-mRNA

• Soon after the transcription and the 5’ cap formation several functions of spliceosome acts on the PO4rylated tail of the RNA polymerase

1- Pre-mRNA coming from RNA polymerase keeps the track of intron and exons

• The 5’ snRNP with only one 3’splice site

•This helps to prevent wrong exon skipping

2- Exon definition hypothesis

• RNA synthesis proceeds with SR proteins act like a component of spliceosome

• They mark 3’ and 5’ splice site starting from 5’ end of RNA

• This involves U1 snRNA, mark the exon boundary and U2AF, help to specify other

snRNPs splice a small fraction of Intron sequences

• A small set of snRNPs that direct spliceosome recognizes only specific set of DNA sequences, AT-AC spliceosome

• Another variation of splicing mechanism exits called as trans-splicing

Trypanosomes produce all their mRNA in this way, however few nematode mRNA are produce by transplicing

RNA splicing and Plasticity

• When a mutation occurs in a nucleotide seq critical for splicing of a particular intron, it did not splice that intron

• The exon will be skipped

• New pattern of splicing comes inaction and creates a cryptic junctions and picks out the best pattern of splice junctions

• If the present one got mutated it will seek out a new one having best pattern

RNA-Processing Enzymes Generate the 3’ end of Eucaryotic mRNAs

• RNA polymerase continues its movement along gene, the spliceosome on the RNA and cut the intron and axon boundaries

• The long C-terminal tail of the RNA polymerase make sure that all the components for the splicing should be present on the RNA

•CstF (Cleavage stimulation factor) and CPSF (cleavage and polyadenylation specificity factor) travel with the RNApolymerase II and transferred to 3’ end processing seq on an RNA mol emerges from the enzyme

Polyadenylation


• Subunits of CPSF are associated with transcription factor TFIID

• During transcription initiation these subunits moves on to RNA polymerase tail

• Once RNA mols comes from the polymerase it will accompanied by the binding proteins to form 3’endof mRNA. 1st RNA cleaved by this

• Next the enzyme called poly-A polymerase adds, one time 200 nt A nt to the 3’end to create a cut

• 5’ and 3’ end has been formed by ATP mol


• Two theories for RNA polymerase processivity loss

1- transfer of 3’ end processing factors from RNA polymerase to RNA causes a conformational changes in the polymerase

2- Lack of 5’ cap of the RNA that arises form polymerase might signals to eh enzyme to terminate transcription

Transport of Eukaryote mRNA from the Nucleus

Nuclear Pore Complex

1- They are aqueous channels in the nuclear membranes that directly connect nucleoplasm and cytosol

2- Small mols < 50,000 daltons can diffuse freely through them

3- Macromols signals cells for import and exportpolymerase or mRNA respectively

Export-ready mRNA molecule

Nuclear Pore Complex

1- They are aqueous channels in the nuclear membranes that directly connect nucleoplasm and cytosol

2- Small mols < 50,000 daltons can diffuse freely through them

3- Macromols signals cells for import and exportpolymerase or mRNA respectively

4- Only useful RNA exported out via pores

5- hnRNPs (heterogenous nuclear ribonuclear proteins, 30 of them in humans)are present in abundant on the pre-mRNA

6- Some are useful in removing hairpin helices from the RNA



7- Besides histones, hnRNP proteins are most abundant in the cell nucleus

8- These proteins help to distinguished b/w mature and unprocessed mRNA

Noncoding RNAs synthesis: rRNA

• Few % of dry cell weight is RNA

•The most abundant RNA in the cell is rRNA ie 80%

• 3-5 % mRNA

• RNA polymerase I (structually similar to II) produces rRNA

• RNA polymerase I have no C-terminal ie why they are neither capped or polyadenylated

• Ribosome are final gene products and a growing cell must synthesize approx. 10 million copies of each type of rRNA in each cell generation to construct its 10 million ribosomes


Eukaryotic rRNAs

• 4 types of rRNAs, one on each copy of ribosome

• 3 of four are 18S, 5.8S and 28S

•5.8S is synthesized from a separate cluster of genes by a different polymerase

• Many chemical modifications occur in the 13,000-nucleotide-long precursor r RNA before the rRNA are cleaved out of it and assembled into ribosomes

• Chemical modifications includes 100 methylations of the 2’OH positions on nt sugars and 100 isomerizations of uridine nt to pseudouridine


Modifications:

• It is made at specific position in the prescursor rRNA

• These positions are specified by several hundred “guide RNAs” which locate themselves via bp to the precursor rRNA thereby move RNA-modifying enzyme to the appropriate position

• Other guide RNAs promote cleavage of the precursor rRNAs in to the mature rRNAs probably by causing conformational changes in the precurosor rRNA

• They all belong to theRNA class of small nucleolar RNAs (snoRNAs)

Nucleolus: A Ribosomal-Producing Factory

• Nucleolus is the rRNA processing site and their assembly into ribosome

• It is large aggregate of macromolecules, including the rRNA genes themselves, precursor RNAs, mature rRNAs, rRNA processing enzymes, snoRNPs, ribosomal protein subunits and partly assembled ribosomes

• An eg: The U6 snRNA is chemically modified by snoRNAs in the nucleolous before its final assembly there into the U6 snRNP

• Telomerase and signal recognition particle are said to be synthesized in nucleolus

• Nucleolus size changes with the type of cells

The Nucleus

• Contains GEMS (Gemini of coiled bodies) and speckles (interchromatin granule clusters)

• GEMS and speckles are paired in the nucleus

•This is the site where snRNAs and snoRNAs undergo their final modifications and assemble with proteins

• They are the Cajal/GEMS site where snRNPs are recycled and their RNAs are “reset” after rearrangements that occur during splicing of pre-mRNA

The Nucleus

• Interchromatin granule clusters have been proposed to be stock piles of fully mature snRNPs that are ready to be used in splicing of pre-mRNA

• GEMS contain SMN (survival motor neurons) proteins. Mutations in GEMS gene encoding for this protein can cause the spinal muscular atrophy

• In this disease subtle defects in snRNPs assembly and subsequent splicing of pre-mRNA takes place

• RNA splicing take place at various location in chromosome bc splicing is co-transcriptional

The Nucleus

• But when the same region become transcriptionally active, they relocate towards the interior of the nucleus, which is richer in the components required for mRNA synthesis

• Mammalian cells express 15,000 genes so transcription and RNA splicing must take place at several thousands sites in the nucleus

The genetic Codon

Translation: Clover Leaf tRNA

Unusual nucleotides found in the tRNA

Amino acid Activation

Aminoacyl-tRNA linkage

Translation of the genetic code

Amino acid incorporation

Hydrolytic RNA Editing

Comparison of the structures of procaryotic and eucaryotic ribosomes

A Ribosome and binding sites

RNA-binding sites in the ribosome

Translating an mRNA molecule

Elongation of a polypeptide chain

Mechanism of Peptidyl transferase activity present in the large ribosomal subunit

Initiation phase of protein synthesis in eucaryotes

Final phase of protein synthesis

A polyribosome

The translational frameshift that produces the reverse transcriptase and integrase of a retrovirus

Inhibitors of Protein or RNA Synthesis

Inhibitor Specific Effect

Acting Only on Procaryotes*

Tetracycline blocks binding of aminoacyl-tRNA to A-site of ribosome

Streptomycin prevents the transition from initiation complex to chain-elongating ribosome and also causes miscoding

Chloramphenicol blocks the peptidyl transferase reaction on ribosomes

Erythromycin blocks the translocation reaction on ribosomes

Rifamycin blocks initiation of RNA chains by binding to RNA polymerase (prevents RNA synthesis)

Acting on Procaryotes and Eucaryotes

Puromycin causes the premature release of nascent polypeptide chains by its addition to growing chain end

Actinomycin D binds to DNA and blocks the movement of RNA polymerase (prevents RNA synthesis)

Acting Only on Eucaryotes

Cycloheximide blocks the translocation reaction on ribosomes

Anisomycin blocks the peptidyl transferase reaction on ribosomes

a-Amanitin blocks mRNA synthesis by binding preferentially to RNA polymerase II

*. The ribosomes of eucaryotic mitochondria (and chloroplasts) often resemble those of procaryotes in their sensitivity to

inhibitors. Therefore, some of these antibiotics can have a deleterious effect on human mitochondria.

Protein folding

Hsp70 family of molecular chaperones

Hydrophobic regions of a protein: Protein Quality control

Ubiquitin regulated degradation of the proteins

Triggering sister chromatid separation

Retroviruses

Retroviruses are infectious particles consisting of an RNA genome packaged in a protein capsid, surrounded by a lipid envelope. This lipid envelope contains polypeptide chains including receptor binding proteins which link to the membrane receptors of the host cell, initiating the process of infection.Retroviruses contain RNA as the hereditary material in place of the more common DNA. In addition to RNA, retrovirus particles also contain the enzyme reverse transcriptase (or RTase), which causes synthesis of a complementary DNA molecule (cDNA) using virus RNA as a template.

When a retrovirus infects a cell, it injects its RNA into the cytoplasm of that cell along with the reverse transcriptase enzyme. The cDNA produced from the RNA template contains the virally derived genetic instructions and allows infection of the host cell to proceed. The virus that causes AIDS (acquired immune deficiency syndrome) is a retrovirus. It is called HIV for human immunodeficiency virus.

Exogenous

The following genera are included here:

• Genus Alpharetrovirus; type species: Avian leukosis virus; others include Rous sarcoma virus • Genus Betaretrovirus; type species: Mouse mammary tumor virus• Genus Gammaretrovirus; type species: Murine leukemia virus; others include Feline Leukemia virus • Genus Deltaretrovirus; type species: Bovine leukemia virus; others include the cancer-causing Human T-lymphotropic virus• Genus Epsilonretrovirus; type species: Wall eye dermal sarcoma virus• Genus Lentivirus; type species: Human immunodeficiency virus 1: others include Simian, Feline immunodeficiency viruses • Genus Spumavirus; type species: Simian Foamy virusThese were previously divided into three subfamilies

Biogenesis of RTs

Retroviruses can be subdivided in to several groups on the basis of Genus

• All grps carry the gag, pol and env coding domains

• Whereas, the complex retroviruses lentivirus and delta viruses carry additional genes encoding for the regulatory proteins

• Reverse transcriptase sequences reside within the pol coding domain. They share similar activities in all types of viruses

• But these transcriptase can be differ in structure and subunit composition, molecular weights, catalytic activity, biochemical, biophysical characteristics and sensitivity to different inhibitors

• All retroviruses, the viral pol gene encodes the Pol precursor protein that consists both reverse transcriptase and integrase proteins

• Fused protein is synthesized as part of a larger Gag-Pro-Pol polyprotein, from which the mature reverse transcriptase and integrase protein are cleaved out during virus assembly by the viral protease

• The Gag and Gag-Pro-Pol polyproteins are translated from identical mRNA but in Gag-Pro-Pol there is either a termination suppression or a frameshift by 1 nt. This regulates the ratio of Gag to Gag –Pro-Pol synthesis

• In some retroviruses like lentivirus there is only 1 nt frameshift takes place in the same reading frame which brings same equimolar ratios of reversetranscriptase, PR and Integrase. This ratio is 20 fold less than the Gag protein.

Some basic features shared by all retroviruses

Alpharetrovirus (ASLV)

• There is 1 nt shift in the reading frame before the pol gene

• Due to this pro gene in the same reading frame as Gag, here Gag is synthesized as a part of the Gag polyprotein

• In this the ratio of viral protease (encoded by the pro gene) to Pol is much higher compared to lentiviruses

Mouse mammary tumor virus (MMTV, betaretrovirus)

• There is 2 consecutive frameshift take place in the reading frame.

• This type of shifting also occurs in T-cell leukemia virus-1 (HTLV-1), bovine leukemia virus (BLV) (both are deltaretroviruses)

• In this pro gene in its own reading frame is in a 1 nt shift relative to gag and the pol gene is in a 1 nt shift relative to pro gene

• In epitomizing gammaretroviruses there is an entirely different independent-frameshift arrangement takes place

• In this pol and pro genes are in the same reading frames as gag, but are separated by a stop codon

Some basic features of retroviruses

• The synthesis of Pro and Pol proteins is a product of an in-frame read-through suppression of the termination codon at the end of the gag gene.

• Frame shifting and read through have probably evolved as strategies to provide proper ratios of Gag, Gag-Pro and Gag-Pro-Pol polypeptides in the infected cell

• Spumaviruses (foamy viruses) are very different from other retroviruses in which reverse transcriptase is generated from a separate spliced mRNA instead of Gag-Pro-Pol polypeptide precursor

• This also include infectous dsDNA particles, who are similar to full length cDNA

• Alpharetroviruses, such as ASLV β subunit has a fused reverse transcriptase-integrase polypeptide while α subunit lacks integrase sequences having RNase H domain

• Gammaretroviruss or betaretroviruses have monomeric reverse transcriptase, containing DNA polymerase and RNase H domains

• Their N-terminal contain in some residues upstream of viral protease protein

Some basic features of retroviruses

Biogenesis of Reverse Transcriptase

Retroviruses Practicality

Gene therapyGammaretroviral and lentiviral vectors for gene therapy have been developed that mediate stable genetic modification of treated cells by chromosomal integration of the transferred vector genomes.

CancerRetroviruses that cause tumor growth include Rous sarcoma virus and mouse mammary tumor virus. Cancer can be triggered by proto-oncogenes that were mistakenly incorporated into proviral DNA or by the disruption of cellular proto-oncogenes.

• A retrovirus is an RNA virus that is replicated in a host cell via the enzyme reverse transcriptase to produce DNA from its RNA genome

• The DNA is then incorporated into the host's genome by an integrase enzyme. The virus thereafter replicates as part of the host cell's DNA

• Retroviruses are enveloped viruses that belong to the viral family Retroviridae

• The virus itself stores its nucleic acid in the form of a +mRNA (including the 5'cap and 3'PolyA inside the virion) genome and serves as a means of delivery of that genome into cells it targets as an obligate parasite, and constitutes the infection

• Once in the host's cell, the RNA strands undergo reverse transcription in the cytosol and are integrated into the host's genome, at which point the retroviral DNA is referred to as a provirus. It is difficult to detect the virus until it has infected the host

Retroviruses

Structure• Virions of retroviruses consist of enveloped particles about 100 nm in diameter. • The virions also contain two identical single-stranded RNA molecules 7-10 kilobases (kb) in length. • Although virions of different retroviruses do not have the same morphology or biology, all the virion components are very similar

The main virion components are:

Envelope composed of a protein capsid, which is obtained from the host plasma membrane during budding process.

RNA: consists of a dimer RNA. It has a cap at 5' end and polyadenyle at 3' end. • The RNA genome also has terminal noncoding regions, which are important in replication, and internal regions that encode virion proteins for gene expression. • The 5' end includes four regions, which are R, U5, PBS, and L. R region is a short repeated sequence at each end of the genome during the reverse transcription in order to ensure correct end-to-end transfer in growing chain.

Retroviruses

The main virion components

RNA:•U5, on the other hand, is a short unique sequence between R and PBS. PBS (primer binding site) consists of 18 bases complementary to 3' end of tRNA primer

• L region is an untranslated leader region that gives signal for packaging of genome RNA. The 3' end includes 3 regions, which are PPT (polypurine tract), U3, and R • PPT is primer for plus-strand DNA synthesis during reverse transcription

• U3 is a sequence between PPT and R, which has signal that provirus can use in transcription. R is the terminal repeated sequence at 3' end.

Proteins: consisted of gag proteins, protease (PR), pol proteins and env proteins • Gag proteins are major components of the viral capsid, which are about 2000-4000 copies per virion

• Protease is expressed differently in different viruses, It functions in proteolytic cleavages during virion maturation to make mature gag and pol proteins

• Pol proteins are responsible for synthesis of viral DNA and integration into host DNA after infection, Finally, env proteins play role in association and entry of virion into the host cell

When retroviruses have integrated their own genome into the germ line, their genome is passed on to a following generation

• These endogenous retroviruses (ERVs), contrasted with exogenous ones, now make up 5-8% of the human genome

• Most insertions have no known function and are often referred to as “junk DNA“

• However, many endogenous retroviruses play important roles in host biology, such as control of gene transcription, cell fusion during placental development in the course of the germination of an embryo, and resistance to exogenous retroviral infection

• Endogenous retroviruses have also received special attention in the research of immunology-related pathologies, such as autoimmune disease like multiple sclerosis, although endogenous retroviruses have not yet been proven to play any causal role in this class of disease

Multiplication

• While transcription was classically thought to only occur from DNA to RNA, reverse transcriptase transcribes RNA into DNA

• The term "retro" in retrovirus refers to this reversal (making DNA from RNA) of the central dogma of molecular biology

• Reverse transcriptase activity outside of retroviruses has been found in almost all eukaryotes, enabling the generation and insertion of new copies of retrotransposons into the host genome

• It is important to note that a retrovirus must "bring" its own reverse transcriptase in its capsid, otherwise it is unable to utilize the enzymes of the infected cell to carry out the task, due to the unusual nature of producing DNA from RNA.

Multiplication

Multiplication

• Industrial drugs that are designed as protease and reverse transcriptase inhibitors can quickly be proved ineffective because the gene sequences that code for the protease and the reverse transcriptase can undergo many substitutions

•These substitutions of nitrogenous bases, which make up the DNA strand, can make either the protease or the reverse transcriptase difficult to attack

•The amino acid substitution enables the enzymes to evade the drug regiments because mutations in the gene sequences can cause physical or chemical change, which makes them harder to detect by the drug

•When the drugs that are supposed to attack enzymes, such as protease, are designed, the manufacturers target specific sites on the enzyme

•One way to attack these targets can be through hydrolysis of molecular bonds, which means that the drug will add molecules of H2O (water) to specific bonds. By adding molecules of water at a site on the virus, the drug breaks the previous bonds that were linked to each other

• If several of these breaks occur, the result can lead to lysis, the death of the virus.Because reverse transcription lacks the usual proofreading of DNA replication, a retrovirus mutates very often. This enables the virus to grow resistant to antiviral pharmaceuticals quickly, and impedes the development of effective vaccines and inhibitors for the retrovirus.

GenesRetrovirus genomes commonly contain these three open reading frames that encode for proteins that can be found in the mature virus:Group specific antigen (gag) codes for core and structural proteins of the virus; Polymerase (pol) codes for reverse transcriptase, protease and integrase; and, envelop (env) codes for the retroviral coat proteins.

ProvirusThis DNA can be incorporated into host genome as a provirus that can be passed on to progeny cells. The provirus DNA is inserted at random into the host genome. Because of this, it can be inserted into oncogenes. In this way some retroviruses can convert normal cells into cancer cells. Some provirus remains latent in the cell for a long period of time before it is activated by the change in cell environment.

DevelopmentStudies of retroviruses led to the first demonstrated synthesis of DNA from RNA templates, a fundamental mode for transferring genetic material that occurs in both eukaryotes and prokaryotes. It has been speculated that the RNA to DNA transcription processes used by retroviruses may have first caused DNA to be used as genetic material. In this model, the RNA worlds hypothesis, cellular organisms adopted the more chemically stable DNA when retroviruses evolved to create DNA from the RNA templates. Retroviruses are proving to be valuable research tools in molecular biology and have been used successfully in gene delivery systems.

EndogenousMain article: endogenous retrovirusEndogenous retroviruses are not formally included in this classification system, and are broadly classified into three classes, on the basis of relatedness to exogenous genera:Class I are most similar to the gammaretroviruses Class II are most similar to the betaretroviruses and alpharetroviruses Class III are most similar to the spumaviruses Group VIAll members of Group VI use virally encoded reverse transcriptase, an RNA-dependent DNA polymerase, to produce DNA from the initial virion RNA genome. This DNA is often integrated into the host genome, as in the case of retroviruses and pseudoviruses, where it is replicated and transcribed by the host.Group VI includes:Family MetaviridaeFamily PseudoviridaeFamily Retroviridae - Retroviruses, e.g. HIV

Group VII

Both families in Group VII have DNA genomes contained within the invading virus particles. The DNA genome is transcribed into both mRNA, for use as a transcript in protein synthesis, and pre-genomic RNA, for use as the template during genome replication. Virally encoded reverse transcriptase uses the pre-genomic RNA as a template for the creation of genomic DNA.Group VII includes:Family Hepadnaviridae - e.g. Hepatitis B virus Family Caulimoviridae - e.g. Cauliflower mosaic virus

TreatmentMain article: Antiretroviral drugAntiretroviral drugs are medications for the treatment of infection by retroviruses, primarily HIV. Different classes of antiretroviral drugs act at different stages of the HIV life cycle. Combination of several (typically three or four) antiretroviral drugs is known as highly active anti-retroviral therapy (HAART).

Treatment of Veterinary RetrovirusesFeline Leukemia Virus and Feline immunodeficiency virus infections are treated with biologics, including Lymphocyte T-Cell Immune Modulator (LTCI)

• RTN is initiated after the association of RT with genomic viral RNA and a primer. The primer is required for copying the template RNA by all RTs

• However in most cases, DNA synthesis is initiated from a transfer RNA (tRNA) primer, supplied by the host cell

• A tRNA lys3 is used by lentivruses and betaretroviruses

• A tRNA pro is used by gammaretroviruses and tRNA trp is used by alpharetroviruses

Reverse Transcription

Process in class VI viruses Class VI viruses ssRNA-RT, also called the retroviruses are RNA reverse transcribing viruses with a DNA intermediate. Their genomes consist of two molecules of positive sense single stranded RNA with a 5’ cap and 3’ polyadenylated tail. Examples of retroviruses include Human Immunodeficiency Virus (HIV) and Human T-Lymphotropic virus (HTLV). Creation of double-stranded DNA occurs in the cytosol as a series of steps

Reverse transcription

Process in class VI viruses

RT in HIV

HIV Virus

Foamy virus

Different types of retroviruses

Post Translational modifications

1- Most of the proteins that are translated from mRNA undergo chemical modifications before becoming functional in different body cells

2- Expression of proteins is important in diseased conditions. Post translational modifications play an important part in modifying the end product of expression and contribute towards biological processes and diseased conditions. The amino terminal sequences are removed by proteolytic cleavage when the proteins cross the membranes. These amino terminal sequences target the proteins for transporting them to their actual point of action in the cell.

Types of Protein Post Translational Modifications

1- Glycosylation: Many proteins, particularly in eukaryotic cells, are modified by the addition of

carbohydrates, a process called glycosylation. Glycosylation in proteins results in addition of a glycosyl

group to either asparagine, hydroxylysine, serine, or threonine.

2- Acetylation: the addition of an acetyl group, usually at the N-terminus of the protein.

3- Alkylation: The addition of an alkyl group (e.g. methyl, ethyl).

4- Methylation: The addition of a methyl group, usually at lysine or arginine residues.

5- Biotinylation: Acylation of conserved lysine residues with a biotin appendage.

6- Glutamylation: Covalent linkage of glutamic acid residues to tubulin and some other proteins.

7- Glycylation: Covalent linkage of one to more than 40 glycine residues to the tubulin C-terminal tail of

the amino acid sequence.

8- Isoprenylation: The addition of an isoprenoid group (e.g. farnesol and geranylgeraniol).

9- Lipoylation: The attachment of a lipoate functionality.

10- Phosphopantetheinylation: The addition of a 4'-phosphopantetheinyl moiety from coenzyme A, as in

fatty acid, polyketide, non-ribosomal peptide and leucine biosynthesis.

11- Phosphorylation: the addition of a phosphate group, usually to serine, tyrosine, threonine or histidine.

12- Sulfation: The addition of a sulfate group to a tyrosine.

13- Selenation: C-terminal amidation

Glycosylation

1- Glycosylation is the enzymatic process that links saccharides to produce glycans, attached to proteins, lipids, or other organic molecules.

2- This enzymatic process produces one of the fundamental biopolymers found in cells (along with DNA, RNA, and proteins).

3- Glycosylation is a form of co-translational and post-translational modification. Glycans serve a variety of structural and functional roles in membrane and secreted proteins. The majority of proteins synthesized in the rough ER undergo glycosylation.

4- It is an enzyme-directed site-specific process, as opposed to the non-enzymatic chemical reaction of glycation.

Five classes of glycans are produced:

1- N-linked glycans attached to a nitrogen of asparagine or arginine side chains;

2- O-linked glycans attached to the hydroxy oxygen of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline side chains, or to oxygens on lipids such as ceramide;

3- phospho-glycans linked through the phosphate of a phospho-serine;

4- C-linked glycans, a rare form of glycosylation where a sugar is added to a carbon on a tryptophan side chain;

5- glypiation, which is the addition of a GPI anchor that links proteins to lipids through glycan linkages.

1- Some proteins do not fold correctly unless they are glycosylated first

2- Polysaccharides linked at the amide nitrogen of asparagine in the protein confer stability on some secreted glycoproteins

3- Experiments have shown that glycosylation in this case is not a strict requirement for proper folding, but the unglycosylated protein degrades quickly.

4- Glycosylation may play a role in cell-cell adhesion

Importance of Glycosylation

1- N-linked glycosylation is important for the folding of some eukaryotic proteins. The N-linked glycosylation process occurs in eukaryote and widely in archaea, but very rarely in bacteria

2- Eukaryotes, most N-linked oligosaccharides begin with addition of a 14-sugar precursor to the asparagine in the polypeptide chain of the target protein. The structure of this precursor is common to most eukaryote, and contains 3 glucose, 9 mannose, and 2 N-acetylglucosamine molecules

N-linked Glycosylation

5- Mature glycoproteins may contain a variety of oligomannose N-linked oligosaccharides containing between 5 and 9 mannose residues.

6- Glucose linked to the guanidinium group of arginine in sweet corn amyelogenin is the only reported example of N-linked glycosylation on an amino acid other than asparagine.

N-linked Glycosylation

1- O-linked glycosylation occurs at a later stage during protein processing, probably in the Golgi apparatus

2- This is the addition of N-acetyl-galactosamine to serine or threonine residues by the enzyme UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase (EC 2.4.1.41), followed by other carbohydrates (such as galactose and sialic acid)

3- This process is important for certain types of proteins such as proteoglycans, which involves the addition of glycosaminoglycan chains to an initially unglycosylated proteoglycan core protein

Importance: One function involves secretion to form components of the extracellular matrix, adhering one cell to another by interactions between the large sugar complexes of proteoglycans. The other main function is to act as a component of mucosal secretions, and it is the high concentration of carbohydrates that tends to give mucus its "slimy" feel.

O-linked glycosylation and O-N-acetylgalactosamine (O-GalNAc)

O-fucose

1- O-fucose is added between the second and third conserved cysteines of EGF-like repeats in the Notch protein.

2- In the case of EGF-like repeats, the O-fucose may be further elongated to a tetrasaccharide by sequential addition of N-acetylglucosamine (GlcNAc), galactose, and sialic acid

3- For Thrombospondin repeats, may be elongated to a disaccharide by the addition of glucose.

O-glucose

1- O-glucose is added between the first and second conserved cysteines of EGF-like repeats in the Notch protein, and possibly other substrates by protein:O-glucosyltransferase (Poglut).

2- The O-glucose modification appears to be necessary for proper folding of the EGF-like repeats of the Notch protein, and increases secretion of this receptor.

O-N-acetylglucosamine

Recently, O-GlcNAc was reported to occur between the fifth and sixth conserved cysteines in some EGF-like repeats from the Notch protein

O-mannose

1- During O-mannosylation, a mannose residue is transferred from mannose-p-dolichol to a serine/threonine residue in secretory pathway proteins.

2- O-mannosylation is common to both prokaryotes and eukaryotes

Collagen Glycosylation

1- Many lysines in collagen are hydroxylated to form hydroxylysine, and many of these hydroxylysines are then glycosylated by the addition of galactose.

2- This galactose monosaccharide can then be further elongated by the addition of a glucose.

3- This glycosylation is required for the proper functioning of collagen. Glycosylation of hydroxlysine occurs in the ER.

Hydroxyproline Glycosylation

Proline is also hydroxylated in collagen, however, no glycosylation occurs here as the hydroxyprolines are necessary for hydrogen bonding in the collagen triple helix.

Glycosylation of Glycogenin

Liver and muscle glycogenin carries a glucose on a tyrosine side chain. This is the only known example of glycosylated tyrosine in nature.

Glycosylation of Ceramide

Either a galactose or a glucose can be added to a hydroxyl on the lipid ceramide. The glucose can be further elongated to a disaccharide by the addition of a galactose.

Proteoglycans

The large and complex glycans that modify proteoglycans are initiated by addition of xylose to serine.

C-mannosylation

1- A mannose sugar is added to the first tryptophan residue in the sequence W-X-X-W (W indicates tryptophan, X is any amino acid).

2- Thrombospondins are one of the most commonly modified proteins, however this form of glycosylation appears elsewhere as well.

3- This is an unusual modification because the sugar is linked to a carbon rather than a reactive atom like a nitrogen or oxygen.

GPI Anchors (Glypiation)

A special form of glycosylation is the GPI anchor. This form of glycosylation functions to attach a protein to a hydrophobic lipid anchor, via a glycan chain

1- O-GlcNAc is added to serines or threonines by O-GlcNAc transferase.

2- O-GlcNAc appears to occur on most serines and threonines that would otherwise be phosphorylated by serine/threonine kinases.

3- O-GlcNAc addition and removal also appears to be a key regulator of the pathways that are disrupted in diabetes mellitus.

4- The gene encoding the O-GlcNAcase enzyme has been linked to non-insulin dependent diabetes mellitus. It is the terminal step in a nutrient-sensing hexosamine signaling pathway.

O-N-acetylglucosamine (O-GlcNAc)

1- Proteins that are membrane bound or are destined for excretion are synthesized by

ribosomes associated with the membranes of the endoplasmic reticulum (ER).

2- The ER associated with ribosomes is termed rough ER (RER). This class of proteins

all contain an N-terminus termed a signal sequence or signal peptide.

3- The signal peptide is usually 13-36 predominantly hydrophobic residues.

4- The signal peptide is recognized by a multi-protein complex termed the signal

recognition particle (SRP).

5- However, some proteins that are destined for secretion are also further proteolyzed

following secretion and, therefore contain pro sequences.

• Mechanism of synthesis of membrane bound or secreted proteins

Secreted and Membrane-Associated Proteins

1- Most proteins undergo proteolytic cleavage following translation.

2- The simplest form of this is the removal of the initiation methionine.

3- Many proteins are synthesized as inactive precursors that are activated under proper physiological conditions by limited proteolysis.

4- Pancreatic enzymes and other enzymes involved in clotting are examples of the latter.

5- Inactive precursor proteins that are activated by removal of polypeptides are termed proproteins.

6- An example of a preproprotein is insulin. Since insulin is secreted from the pancreas it has a prepeptide.

7- Following cleavage of the 24 amino acid signal peptide the protein folds into proinsulin. 8- Proinsulin is further cleaved yielding active insulin which is composed of two peptide chains linked together through disulfide bonds.

Proteolytic Cleavage

1- Many proteins are modified at their N-termini following synthesis.

2- In most cases the initiator methionine is hydrolyzed and an acetyl group is added to the new N-terminal amino acid.

3- Acetyl-CoA is the acetyl donor for these reactions.

4- Some proteins have the 14 carbon myristoyl group added to their N-termini.

5- The donor for this modification is myristoyl-CoA.

6- This latter modification allows association of the modified protein with membranes.

7- Histone acetylation occurs on the surface of the nucleosome core as part of gene regulation when the histones are acetylated on lysine residues.

8- Acetylation brings in a negative charge that acts to neutralize the positive charge on the histones,

9- This decreases the interaction of the N termini of histones with the negatively charged phosphate groups of DNA.

Acetylation

1- Post-translational methylation of proteins occurs on nitrogens, oxygens, Imidazole ring of histidine, Guanadino moiety of Arginine, R-grp amides of Glutamate and Aspartate

2- The activated methyl donor is S-adenosylmethionine (SAM).

3- The most common methylations are on the ε-amine of lysine residues.

4- Methylation of lysine residues in histones in DNA is an important regulator of chromatin structure and consequently of transcriptional activity.

5- Recent findings indicate that methylation of lysine residues affects gene expression not only at the level of chromatin, but also by modifying transcription factors.

Methylation

1- Post-translational phosphorylation is one of the most common protein modifications that occurs in animal cells.

2- The vast majority of phosphorylations occur as a mechanism to regulate the biological activity of a protein and as such are transient.

3- In other words a phosphate (or more than one in many cases) is added and later removed.

4- Physiologically relevant examples are the phosphorylations that occur in glycogen synthase and glycogen phosphorylase in hepatocytes in response to glucagon release from the pancreas.

5- Phosphorylation of synthase inhibits its activity, whereas, the activity of phosphorylase is increased. These two events lead to increased hepatic glucose delivery to the blood.

6- In animal cells serine, threonine and tyrosine are the amino acids subject to phosphorylation.

Phosphorylation

1- Sulfate modification of proteins occurs at tyrosine residues such as in fibrinogen and in some secreted proteins (eg gastrin).

2- The universal sulfate donor is 3'-phosphoadenosyl-5'-phosphosulphate

3- Since sulfate is added permanently it is necessary for the biological activity and not used as a regulatory modification like that of tyrosine phosphorylation.

Sulfation

1- Prenylation refers to the addition of the 15 carbon farnesyl group or the 20 carbon geranylgeranyl group to acceptor proteins, both of which are isoprenoid compounds derived from the cholesterol biosynthetic pathway.

2- The isoprenoid groups are attached to cysteine residues at the carboxy terminus of proteins in a thioether linkage (C-S-C).

3- Some of the most important proteins whose functions depend upon prenylation are those that modulate immune responses.

4- These include proteins involved in leukocyte motility, activation, and proliferation and endothelial cell immune functions.

5- It is these immune modulatory roles of many prenylated proteins that are the basis for a portion of the anti-inflammatory actions of the statin class of cholesterol synthesis-inhibiting drugs

Prenylation

1- Modifications of proteins that depend upon vitamin C as a cofactor include proline and lysine hydroxylations and carboxy terminal amidation.

2- The hydroxylating enzymes are identified as prolyl hydroxylase and lysyl hydroxylase.

3- The donor of the amide for C-terminal amidation is glycine. The most important hydroxylated proteins are the collagens.

4- Several peptide hormones such as oxytocin and vasopressin have C-terminal amidation.

Vitamin C-Dependent Modifications

1- Vitamin K is a cofactor in the carboxylation of glutamic acid residues.

2- The result of this type of reaction is the formation of a γ-carboxyglutamate (gamma-carboxyglutamate), referred to as a gla residue.

3- The formation of gla residues within several proteins of the blood clotting cascade is critical for their normal function.

4- The presence of gla residues allows the protein to chelate calcium ions and thereby render an altered conformation and biological activity to the protein.

5- The coumarin-based anticoagulants, warfarin and dicumarol function by inhibiting the carboxylation reaction.

Structure of a gla Residue

Vitamin K-Dependent Modifications

1- Selenium is a trace element and is found as a component of several prokaryotic and eukaryotic enzymes that are involved in redox reactions.

2- The selenium in these selenoproteins is incorporated as a unique amino acid, selenocysteine, during translation.

3- A particularly important eukaryotic selenoenzyme is glutathione peroxidase.

4- This enzyme is required during the oxidation of glutathione by hydrogen peroxide (H2O2) and organic hydroperoxides. Structure of the Selenocysteine

Residue

Selenoproteins

1- Proteins are in a continual state of flux, being synthesized and degraded.

2- In addition, when proteins become damaged they must be degraded to prevent aberrant activities of the defective proteins and/or other proteins associated with those that have been damaged.

3- Proteins that are to be degraded by the proteosome are first tagged by attachment of multimers of the 76 amino acid polypeptide ubiquitin.

4- Many proteins involved in cell cycle regulation, control of proliferation and differentiation, programmed cell death (apoptosis), DNA repair, immune and inflammatory processes and organelle biogenesis have been discovered to undergo regulated degradation via the 26S proteosome.

5- Of clinical significance are the recent findings that deregulation of the functions of the proteosome can contribute to the pathogenesis of various human diseases such as cancer, myeloproliferative diseases, and neurodegenerative diseases.

Ubiquitin and Targeted Protein Degradation

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Basic Processes of Molecular Biology