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Lecture 25
Translation (protein synthesis)
5’3’
From messenger RNA to protein:
The information encoded in the DNA is transferred to messenger RNA and then decoded by the ribosome toproduce proteins.
• Process of making proteinsproteins from info on mRNAmRNA– mRNA travels out of
nucleus to the ribosome, which “reads” the mRNA as a series of 3 letter words called codons
Translation
• Translation is the production of a protein using the information that is coded in the mRNA molecule
• In the cytoplasm a ribosome attaches to one end of the mRNA molecule. using a sequence that is the binding site for the ribosome
Second mRNA base
Fir
st
mR
NA
ba
se
(5 e
nd
)
Th
ird
mR
NA
ba
se
(3 e
nd
)
Genetic Code
The Code
• 64 codons
•Genetic code–redundant but not ambiguous; no codon specifies more than one amino acid (but one amino acid may have >1 codon)
•Codons– must be read in the correct reading frame in order for the specified polypeptide to be produced
Evolution of the Genetic Code
• Genetic code– nearly universal: shared by the simplest bacteria, plants,
fungi and animals
• Genes can be transcribed and translated after being transferred from one species to another
Mechanism of Translation
• Ribosomes- Bind messenger (mRNA)- Attract transfer RNA (tRNA) to mRNA – tRNA covalently linked to specific amino acid (aa-tRNA)
-Complementary basepairs form between mRNA and aa-tRNA (codon-anticodon interactions)
-Enzyme in ribosome catalyzes peptide bond between amino acids- -> polypeptide chain grows
Transfer RNA
• Transfer RNA molecules are short RNAs that fold into a characteristic cloverleaf pattern. Some of the nucleotides are modified to become things like pseudouridine and ribothymidine.
• Each tRNA has 3 bases that make up the anticodon. These bases pair with the 3 bases of the codon on mRNA during translation.
• Each tRNA has its corresponding amino acid attached to the 3’ end. A set of enzymes, the “aminoacyl tRNA synthetases”, are used to “charge” the tRNA with the proper amino acid.
• Some tRNAs can pair with more than one codon. The third base of the anticodon is called the “wobble position”, and it can form base pairs with several different nucleotides.
Generalized tRNA tRNA structure
~ 80 nt long
Two-dimensional structure
Three-dimensional structure
Chemistry of tRNA Charging with Amino Acid
tRNAs are activated by amino-acyl tRNA synthetases
Amino acid Aminoacyl-tRNAsynthetase (enzyme)
Pyrophosphate
Phosphates
tRNA
AMP
Aminoacyl tRNA(an “activatedamino acid”)
Amino-acyl tRNA synthetases:
One synthetase for each amino acida single synthetase may recognize multiple tRNAsfor the same amino acid
Two classes of synthetase. Different 3-dimensional structuresDiffer in which side of the tRNA they recognize and how they bind ATP
Class I - monomeric, acylates the 2’OH on the terminal riboseArg, Cys , Gln, Glu, Ile, Leu, Met, Trp Tyr, Val
Class II - dimeric, acylate the 3’OH on the terminal riboseAla, Asn, Asp, Gly, His, Lys, Phe, Ser, Pro, Thr
Structure of an amino acyl-tRNA synthetase bound to a tRNA
One mechanism for maintaining high fidelity of proteinsynthesis is the high fidelity of aa-tRNA synthetases
Two levels of control to ensure that the proper amino acidis incorporated into protein: 1) Charging of the proper tRNA
2) Matching the tRNA to the messenger RNA
Incorporation of amino acids into polypeptide chains
Incorporation of the correct amino acyl-tRNA is determinedby base-pairing interactions between the anticodon of the tRNA and the messenger RNA
Protein synthesis occurs on ribosomes
Protein synthesis occurs on ribosomes
Ribosomes
tRNAmolecules
Exit tunnelGrowingpolypeptide
Largesubunit
mRNA 3
Computer model of functioning ribosome
Smallsubunit
5
E P A
• Facilitate specific coupling of anticodons with codons• Ribosomal structure
– Two ribosomal subunits (large and small) • Made of proteins (ribosomal proteins) and ribosomal RNA (rRNA)
Form binding sites for mRNA and aa-tRNA
Structure of a ribosome
and mitochondria
S=Sedimentation coefficient
Ribosome Assembly
The proteins of each ribosomal subunitare organized aroundrRNA molecules
16S rRNA
23S rRNA secondary structure
Ribosome Assembly: takes place largely in a specialized domain ofthe nucleus, the nucleolus
In the nucleolus, RNA polymerase I transcribes the rDNA repeatsto produce a 45S RNA precursor
The 45S precursor is processedand cleaved intomature rRNAs andribosomal proteinsthen bind to generatethe large and smallribosomal subunits
3D organization of the eukaryotic large subunit rRNA
Ribosomal Proteins decorate the surface of the ribosome
Large subunit. Grey = rRNA Gold = ribosomal proteins
The ribosomal proteins are important for maintaining the stability and integrity of the ribosome, but NOT for catalysis
ie. the ribosomal RNA acts as a ribozyme
Ribosomal proteins often have extensions that chain into the core of the rRNA structure
Crystal structure of L19 L15 (yellow) positioned in a fragmentof the rRNA (red)
Mitochondrialor Prokaryotic
Eukaryotic 60S subunit 80S ribosome 40S subunit
The large and small subunits come together to form the ribosome
The association of the large and small subunits creates the structural features on the ribosome that are essential for protein synthesis
Three tRNA bindingsites:A site = amino-acyltRNA binding site
P site = peptidyl-tRNAbinding site
E site = exit site
In addition to the APE sites there is an mRNA binding groovethat holds onto the message being translated
There is a tunnel through the large subunit that allows thegrowing polypeptide chain to pass out of the ribosome
Mechanism of protein synthesis
Protein synthesis falls into three stages .
1.initiation-the assembly of a ribosome on an mRNA molecule.
2.elongation-repeated cycles of amino acid addition.
3.termination-the release of the new protein chain.
•All three stages require protein translation factors
prokaryotic eukaryotic functionInitiation factor
IF1 IF3
IF2
eIF3 eIF4c eIF6 eIF4B eIF4F
eIF2B eIF2
eIF5
Bind to ribosome submits
Bind to mRNA
Initiator tRNA delivery
Displacement of other factors
Elongation factor
EF-Tu
EF-Ts
EF-g
eEF1α
eEF1βγ
eEF2
Aminoacyl tRNA delivery
Recycling of EF-Tu or eEF1α
Translocation
Termination factors
RF1, RF2, RF3 eRF Polypeptides Chain release
Initiation is controlled differently in prokaryotic and eukaryotic ribosomes
In prokaryotes a single transcript can give rise to multiple proteins
Initiation
Polyribosomes (exists in prokaryotes and viruses)
• a single mRNA (transcript) is translated by many ribosomes simultaneously
• mRNA+ bound ribosomes= polyribosomes (polysome)
• Allows fast synthesis of many copies a polypeptide
RNA polymerase
DNA
Polyribosome
RNApolymerase
Direction oftranscription
mRNA
0.25 m
DNA
Polyribosome
Polypeptide(amino end)
Ribosome
mRNA (5 end)
Messenger RNAs are translated on polyribosomes
Initiation (prokaryote)
In prokaryotes, initiation requires • the large and small ribosome
subunits,• the mRNA • the initiator tRNA • three initiation factors .
• In prokaryotes, ribosomes bind to specific translation initiation sites. There can be several different initiation sites on a messenger RNA: a prokaryotic mRNA can code for several different proteins. Translation begins at an AUG codon, or sometimes a GUG. The modified amino acid N-formyl methionine is always the first amino acid of the new polypeptide.
• In eukaryotes, ribosomes bind to the 5’ cap, then move down the mRNA until they reach the first AUG, the codon for methionine. Translation starts from this point. Eukaryotic mRNAs code for only a single gene. (Although there are a few exceptions, mainly among the eukaryotic viruses).
• Note that translation does not start at the first base of the mRNA. There is an untranslated region at the beginning of the mRNA, the 5’ untranslated region (5’ UTR).
Initiation
Initiator tRNA
• Methionine is the first amino acids incorporated into a protein chain in both prokaryotes (modified to N-formylmethionine) and eukaryotes.
• Initiator tRNAs are special tRNAs recognizing the AUG (Alternative start codon GUG) start codons in prokaryotes and eukaryotes.
• Initiator tRNAs differ from the one that inserts internal Met residues.
Initiator tRNA formation in E. coli
1. Both initiator tRNA and noninitiator tRNAmet are charged with Met by the same methionyl-tRNA synthetase to give the methionyl-tRNA
2. Only the initiator methionyl-tRNA is modified by transformylase to give N-formylmethionyl-tRNAfmet.
Size comparisons show that the ribosome is large enough to bind tRNAs and mRNA.
Met
GTPInitiator tRNA
mRNA
53
mRNA binding site
Smallribosomalsubunit
Start codon
P site
5 3
Translation initiation complex
E A
Largeribosomalsubunit
GDP
Met
• The initiation process involves first joining the mRNA, the initiator methionine-tRNA, and the small ribosomal subunit. Several “initiation factors”--additional proteins--are also involved. The large ribosomal subunit then joins the complex.
Initiation (prokaryote)
Schematic model showing binding sites on ribosome
P site (Peptidyl-tRNAbinding site)
E site (Exit site)
mRNAbinding site
A site (Aminoacyl-tRNA binding site)
Largesubunit
Smallsubunit
E P A
The assembled ribosome has one exit site and two tRNA-binding sites, which are called A- and P-site, for aminoacyl and peptidyl sites respectively.
Only fMet-tRNAfMet can be used for initiation by 30S subunits; all other aminoacyl-tRNAs are used for elongation by 70S ribosomes.
In prokaryotes, specific sequences in the mRNA aroundthe AUG codon, calledShine-Delgarno (SD) sequences,are recognized by an intiationcomplex consisting of a Metamino-acyl tRNA, Initiation Factors (IFs) and the smallribosomal subunit
GTP hydrolysis by IF2 coincident with release of the IFs and binding of the largeribosomal subunit leads to formation of a completeribosome,on the mRNAand ready to translate.
IF1 and IF3 bind to a free 30S subunits.
IF2 complexed with GTP then bind to the small subunits, forming a complex at RBS.
The initiator tRNA can then bind to the complex at the P site paired with AUG codon.
The 50S subunits can now bind. GTP is then hydrolyzed and IFs are released to give the 70S initiation complex
30S initiation complex
Most of the differences in the mechanism of protein between prokaryotes and eukaryotes occur in the initiation stage, where a greater numbers of eIFs and a scanning process are involed in eukaryotes.
The eukaryotic initiator tRNA does not become N-formylated.
Initiation (eukaryote)
The initiation factor can be grouped to there function as follow
Binding to ribosomal subunits eIF6 eIF3 eIF4c
Binding to the mRNA eIF4B eIF4F eIF4A eIF4E
Involved in initiation tRNA delivery
eIF2 eIF2B
Displace other factors eIF5
Initiation (eukaryote)
In contrast to the events in prokaryotes, initiation involves the initiation tRNA binding to the 40S subuits before it can bind to the mRNA. Eukaryotic mRNA binding to the ribosomes is facilitated by the 7-methyl guanosine cap
Eukaryotic mRNAs have a distinct structure at the 5’ end
Structure of the 7-methyl guanosine cap
The 7me-G cap is requiredfor an mRNA to be translated
In contrast, Eukaryotesuse a scanning mechanismto intiate translation.
Recognition of the AUGtriggers GTP hydrolysisby eIF-2
GTP hydrolysis byeIF2 is a signal forbinding of the largesubunit and beginningof translation
Initiation (eukaryote) Scanning
The eukaryotic 40s ribosome submit complex bind to the 5’cap region of the mRNA and moves along it scanning for an AUG start codon.
Eukaryotic ribosomes migrate from the 5’ end of mRNA to the ribosome binding site, which includes an AUG initiation codon.
Elongation
With the formation of the 70S initiation complex, the elongation cycle can begin.
Elongation involves the three factors, EF-Tu, EF-Ts, EF-G, as well as GTP, charged tRNA and the 70S initiation complex.
The protein synthesis elongation cycle in prokaryotes and eukaryotes is quite similar.
The factors EF-Tu EF-Ts EF-G have direct eukaryotic equivalents called eEF1α eEF1βγ eEF2
Ribosome Association and Initiation of Translation
1. Small ribosomal subunit binds mRNA and special initiator tRNA (met-tRNAi)
(carries the amino acid methionine)
2. Small subunit scans along the mRNA until first start codon (AUG).
3. Initiation factors bring in large subunit
initiator tRNA occupies the P site.
Polypeptide
tRNA withamino acidattached
Ribosome
tRNA
Anticodon
35
mRNA
Aminoacids
Codons
Elongation
- Amino acids are added one by one to the preceding amino acid
-Elongation factors facilitate
- codon recognition
- peptide bond formation
- translocation
Amino end
mRNA
5
3
Growing polypeptide
Next amino acidto be added topolypeptide chain
tRNA
Codons
Schematic model with mRNA and tRNA
E
Ribosome translates 5’ to 3’ on mRNA.Polypeptide chain grows amino end first, carboxyl end last.
The ribosome then moves down one codon. The now-empty tRNA at the P site is displaced off the ribosome
The process is then repeated: the tRNA at the P site holds the peptide chain, and a new tRNA binds to the A site.the peptide chain is transferred onto the amino acid attached to the A site tRNA.the ribosome moves down one codon, displacing the empty P site tRNA and moving the tRNA with the peptide chain from the A site to the P site.
Elongation
Ribosome ready fornext aminoacyl tRNA
mRNA
5
Amino endof polypeptide
E
Psite
Asite
3
2
2 GDP
E
P A
GTP
GTP
GDP
E
P A
E
P A
1. Recognition
2. Peptide bondformation
3. Translocation
The three steps of elongation
1.Charged tRNA is delivered as a complex with EF-Tu and GTP .
2.Peptidyl tranferase (50S ribosomal subunit) makes a peptide bond by joining the two adjacent amino acid without the input of more energy.
3.Translocase (EF-G), with the energy from GTP, moves the ribosome one codon along the mRNA, ejecting the uncharged tRNA and transferred the ribosome peptide from the mRNA.
EF-Tu-Ts exchange cycle
A new tRNA, corresponding to the next codon on the mRNA, binds to the A site. The ribosome catalyzes a transfer of the amino acid from the P site onto the amino acid at the A site, forming a new peptide bond.
Peptide bond formation takes place by reaction between the polypeptide of peptidyl-tRNA in the P site and the amino acid of aminoacyl-tRNA in the A site.
Peptide bond formation is catalyzed by the large subunit rRNA
Translocation
• In bacteria, the discharged tRNA leaves the ribosome via another site, the E site. • In eukaryotes, the discharged tRNA is expelled directly into the cytosol.• EF-G (translocase) and GTP binds to the ribosome, and the discharged tRNA is ejected from the P-site in an energy consuming step.• the peptidyl-tRNA is moved from A-site to P-site and mRNA moves by one codon relative to the ribosome
Proper reading of theanticodon is the secondimportant quality controlstep ensuring accurateprotein synthesis
=EF-1 Elongation factors Introduce a two-step“Kinetic proofreading”
A second elongation factorEF-G or EF-2, drives the translocation of the ribosome along the mRNA
Together GTP hydrolysisby EF-1 and EF-2 help driveprotein synthesis forward
• Occurs when stop codon in mRNA reaches A site of ribosome
• Three codons are called “stop codons”. They code for no amino acid, and all protein-coding regions end in a stop codon.
• A site accepts protein called release factor • When the ribosome reaches a stop codon, there is
no tRNA that binds to it. Instead, proteins called “release factors” bind, and cause the ribosome, the mRNA, and the new polypeptide to separate. The new polypeptide is completed.
• Release factor causes addition of water molecule instead of amino acid
Termination
• Note that the mRNA continues on past the stop codon. The remaining portion is not translated: it is the 3’ untranslated region (3’ UTR).
3
The release factor hydrolyzes thebond between the tRNA in theP site and the last amino acid of thepolypeptide chain. The polypeptideis thus freed from the ribosome.
The two ribosomal subunitsand the other componentsof the assembly dissociate.
Releasefactor
Stop codon(UAG, UAA, or UGA)
5
3
5
3
5
Freepolypeptide
When a ribosome reaches a stopcodon on mRNA, the A site of theribosome accepts a protein calleda release factor instead of tRNA.
Termination
Protein factors called release factors interact with stop codon and cause release of completed polypeptide chain.
RF1 and RF2 recognizes the stop codon with the help of RF3
The release factors make peptidyl transferase transfer the polypeptide to water, and thus the protein is released
Release factors and EF-G: remove the uncharged tRNA and release the mRNA.
Termination (prokaryote)
Termination (eukaryote)
Eukaryotes use only one release factors eRF, which requires GTP,recognize all three termination codons.
Termination codon is one of three (UAG, UAA, UGA) that causes protein synthesis to terminate.
Release Factor is a molecular mimic of a tRNA
eRF1 tRNA
TRANSCRIPTION
RNA PROCESSING
RNAtranscript
5
Exon
NUCLEUS
FORMATION OFINITIATION COMPLEX
CYTOPLASM
3
DNA
RNApolymerase
RNA transcript(pre-mRNA)
Intron
Aminoacyl-tRNAsynthetase
Aminoacid
tRNA
AMINO ACID ACTIVATION
3
mRNA
A
P
E Ribosomalsubunits
5
Growingpolypeptide
E A
Activatedamino acid
Anticodon
TRANSLATION
Codon
Ribosome
Post-Translational Modification• New polypeptides usually fold themselves spontaneously into
their active conformation. However, some proteins are helped and guided in the folding process by chaperone proteins
• Many proteins have sugars, phosphate groups, fatty acids, and other molecules covalently attached to certain amino acids. Most of this is done in the endoplasmic reticulum.
• Many proteins are targeted to specific organelles within the cell. Targeting is accomplished through “signal sequences” on the polypeptide. In the case of proteins that go into the endoplasmic reticulum (ER), the signal seqeunce is a group of amino acids at the N terminal of the polypeptide, which are removed from the final protein after translation.
Targeting Polypeptides to Specific Locations
In eukaryotes, what are the two populations of ribosomes?
Free, soluble in cytosol synthesize soluble proteins
Bound to rER- synthesize secreted or membrane bound proteins
- tagged with signal peptide at amino end
Ribosomes
mRNASignalpeptide
Signal-recognitionparticle(SRP)
SRPreceptorprotein
CYTOSOL
ER LUMEN Translocationcomplex
Signalpeptideremoved
ERmembrane
Protein
Signal peptide targets polypeptides to ERfinal polypeptide destined for secretion or membrane
Is the molecular weight of a secreted protein different than the predicted translation product of its mRNA?
Effect of mutations on gene expression
Any change in the genetic material
What is a mutation?
Types of mutationsPoint: a single nucleotide change
-substitution gcca->gcga
-deletion gcca->gca
-insertion gcca->gacca
• From spontaneous mutations: occur during DNA replication, recombination, or repair
• From mutagens are physical or chemical agents that can cause mutations
Source of Mutations
Wild-type hemoglobin DNA
3 5 53
Mutant hemoglobin DNA
Normal hemoglobin Sickle-cell hemoglobin
What kind of mutation? substitution
Substitutions
• Missense mutations– Change codon to encode a different amino acid
• Nonsense mutations– Change codon to encode a stop codon
nearly always leading to a nonfunctional protein
Missense mutations are more common.
Base-pair substitution
No effect on amino acid sequenceU instead of C
MissenseA instead of G
NonsenseU instead of A
Stop
Amino end
Protein
5 3
Carboxyl end
Stop
Stop
Stop
mRNA
Wild type
Neutral
Change in amino acid
Premature termination
Substitutions
Insertions and Deletions
• Alters reading frame ->frameshift mutation
• Often more devastating than substitutions
Ribosomal Frameshifting
Because translationuses a triplet code,there are three potentialreading frames in each mRNA
As the ribosome translocates, it moves in three nucleotidesteps, ensuring that the frame defined by the AUG is usedthroughout translation
If the ribosome moves 1 or 2 (or 4 or 5) nucleotidesthis produces a frameshift
Many retroviruses induce ribosomal frameshifting in the synthesis of viral proteins
e.g. HIV
Protein synthesis is often regulated at the level of translation initiation
General control of translational initiation
ie. all transcripts of the cell are effected (though the relativeeffect differs between specific mRNAs)
Global downregulation or upregulation can occur in response to various stimuli the most common are
1) Nutrient availabilitylow nutrient (amino acids/carbohydrate) downregulates translation
2) Growth factor signals.stimulation of cell division upregulates translation
General control of translational initiation is exerted through two primary mechanisms.
Control of the phosphorylation of eIF2
Control of the phosphorylation of eIF4 binding proteins
Control of translation by eIF2 phosphorylation
Stimulated byAmino acid deprivation
Control of translation by eIF4E availability
The 7MEG cap binding subunit of eIF4, eIF4E, is sequesteredby eIF4E binding proteins (4E-BPs). The binding of theseproteins is regulated by their phosphorylation state
GrowthFactors
NutrientLimitation
An example of control of specific mRNAs: regulation by iron (Fe):
Ferritin is a cytosolic iron binding protein expressed wheniron is abundant in the cell.
Transferrin receptor is a plasma membrane receptor important for the import of iron into the cytosol.
They are coordinately regulated, in opposite directions, bycontrol of protein synthesis.
Regulation by iron (Fe):
Inhibitors of Protein Synthesis
• Those that affect prokaryotic but not eukaryotic protein synthesis are effective antibiotics
• Streptomycin - an aminoglycoside antibiotic - induces mRNA misreading. Resulting mutant proteins slow the rate of bacterial growth
• Puromycin - binds at the A site of both prokaryotic and eukaryotic ribosomes, accepting the peptide chain from the P site, and terminating protein synthesis
Translation Inhibitors are important antibiotics
Sample questions
• The site of protein synthesis is
• A. Ribosome
• B. Nucleus
• C. Endoplasmic reticulum
• D. Chromosome
Sample questions
• The structure in a bacterium that indicates an active site for protein synthesis is
• A. a chromosome.
• B. a cell membrane,
• C. a flagellum.
• D. a polysome.
Sample questions
• Which of the following is not necessary for protein synthesis to occur, once transcription is completed?
• A. tRNA• B. Ribosomes• C. mRNA• D. DNA
Sample questions
• During the process of translation:
• A. the peptide is ‘passed’ from the tRNA in the P-site to the tRNA in the A-site.
• B. incoming tRNAs must first bind to the E-site.
• C. initiation begins with the binding of the ribosomal SSU to the poly-A tail of the mRNA.
• D. the mRNA is translated by one ribosome at a time.
• The nucleolus of the nucleus is the site where:
• A. RNA processing occurs
• B. rRNA is transcribed and ribosomal subunits are assembled
• C. tRNA are charged with amino acids
• D. mRNA is translated into protein
• The ribosomes are composed of
• A. proteins
• B. RNA
• C. both (a) and (b)
• D. lipids
• Which is required for protein synthesis?
• A. tRNA
• B. mRNA
• C. rRNA
• D. All of these
• In the genetic code there are:
• A. more tRNAs than codons.• B. more codons than amino acids.• C. more nucleotides than codons. • D. the same number of codons and amino acids
• he anticodon of tRNA
• A. binds to rRNA• B. binds to an amino acid• C. binds to the Shine Dalgarno sequence• D. binds to an mRNA codon
• Initiation of eukaryotic translation begins when the:
• A. large and small subunits link together, then bind to the mRNA.
• B. ribosomal small subunit holding an initiator tRNA binds to the 5’ end of mRNA.
• C. ribosome binds to of the start codon and an initiator tRNA enters the ribosome.
• D. initiator tRNA binds to the start codon, followed by binding of the ribosome large subunit.
• On the ribosome, mRNA binds
• A. between the subunits• B. to the large subunit• C. to the small subunit• D. none of these
• The peptidyl transferase reaction occurs
• A. on the large subunit
• B. on the small subunit
• C. between the subunit
• D. none of these
• Ribosomes select the correct tRNAs
• A. based on the aminoacyl group
• B. solely on the basis of their anticodons
• C. depending on their abundance in the cytosol
• D. with the least abundant anticodons
• Which of the following amino acid starts all proteins synthesis?
• A. Glycine
• B. Proline
• C. Thymine
• D. Methionine
• The growing polypeptide chain is released from the ribosomes when
• A. a chain terminating codon is reached
• B. a chain terminating tRNA binds to the ribosome
• C. the 7-methyl guanosine cap is reached
• D. the poly A tail is reached
What is a gene?
• A gene is a region of DNA whose final product is either a polypeptide or an RNA molecule