LECTURE PRESENTATIONSFor CAMPBELL BIOLOGY, NINTH EDITION

Jane B. Reece, Lisa A. Urry, Michael L. Cain, Steven A. Wasserman, Peter V. Minorsky, Robert B. Jackson

© 2011 Pearson Education, Inc.

Lectures byErin Barley

Kathleen Fitzpatrick

Regulation of Gene Expression

Chapter 18

Gene expression: Bacteria vs. Eukaryotes

• prokaryotes and eukaryotes alter gene expression in response to their changing environment

– gene expression = refers to the entire process whereby genetic information is decoded into a protein

• prokaryotes and eukaryotes carry out gene expression in similar ways– transcription using an RNA polymerase – translation using ribosomes

• but there are some differences:– 1. RNA polymerases differ – only one in prokaryotes; 3 in eukaryotes– 2. transcription factors used by eukaryotes– 3. transcription is terminated differently in prokaryotes vs. eukaryotes– 4. ribosomes – bacterial ones are smaller– 5. lack of compartmentalization in bacteria – transcribe and translate at the same time

So what is a gene?• unit of inheritance• located on chromosomes• region of specific nucleotide sequence located along the length of DNA• DNA sequence that codes for a specific sequence of amino acids• BUT: some DNA sequences are NEVER translated

– e.g. rRNA and tRNA are transcribed but not translated into anything• so a gene is a region of DNA that is either

– 1. translated into a sequence of amino acids (polypeptide) functional protein– 2. transcribed into a RNA molecule

So what is a gene?• molecular components of a gene:

– A. coding sequences - eukaryotes have introns within their coding sequence– B. promoter– C. enhancers – found in eukaryotes– D. UTRs – found in eukaryotes– E. poly-adenylation sequence – found within the eukaryotic 3’ UTR

Overview: Conducting the Genetic Orchestra

• genetic and biochemical work in bacteria identified two things– 1. protein-binding regulatory sequences associated with genes– 2. proteins that can bind these regulatory sequences – either activating or repressing

gene expression• these two components underlie the ability of both prokaryotic and eukaryotic cells

to turn genes on and off

Bacteria often respond to environmental change by regulating transcription

• natural selection has favored bacteria that produce only the products needed by that cell

• bacteria regulate the production of enzymes by feedback inhibition or by gene regulation

• gene expression in bacteria is controlled by the operon model

Precursor

Feedbackinhibition

Enzyme 1

Enzyme 2

Enzyme 3

Tryptophan

(a) (b)Regulation of enzymeactivity

Regulation of enzymeproduction

Regulationof geneexpression

trpE gene

trpD gene

trpC gene

trpB gene

trpA gene

Operons: Definitions & Concepts• bacteria group functionally related genes so they can be under coordinated

control by a single “on-off regulatory switch” • the regulatory “switch” is a segment of DNA called an operator

– binding sites for transcription factors that help RNA polymerase II bind the nearby promoter

• the operator can be controlled by proteins or nutrients– e.g. can be switched off by a protein called a repressor– repressor prevents gene transcription - binds to the operator and blocks RNA

polymerase binding to the promoter– repressor is the product of a separate regulatory gene– repressor can be in an active or inactive form, depending on the presence of other

molecules• co-repressor is a molecule that cooperates with a repressor protein to switch

an operon off– e.g. the amino acid tryptophan

Operons: Definitions & Concepts

• operon = the entire stretch of DNA that includes the operator, the promoter, and the genes that the promoter controls– the transcription of the downstream genes is polycistronic– produces one long piece of mRNA containing multiple

transcription units

• two kinds – “On” and “Off” operons

Promoter

trp operon

Genes of operon

Operator

mRNA 5Start codon Stop codon

trpE trpD trpC trpB trpA

E D C B A

Polypeptide subunits that make upenzymes for tryptophan synthesis

Repressible and Inducible Operons: Two Types of Negative Gene Regulation

• OFF operon = repressible operon is one that is usually on but is turned OFF by a repressor– e.g. the trp operon is a repressible operon

• ON operon = inducible operon is one that is usually off but is turned ON by an inducer– e.g. lac operon is an inducible operon

The trp Operon: Repressible Operons• E. coli can synthesize the amino acid tryptophan when it is absent from the

growth media• by default the trp operon is on and the genes for tryptophan synthesis are

transcribed• comprised of the:

– 1. operator – capable of binding a repressor protein– 2. genes of the operon – for synthesizing tryptophan when it is missing from the

growth media

• plus a regulatory gene = trpR– expressed whether tryptophan is absent or present

Promoter

DNA

Regulatory gene

mRNA

trpR

5

3

Protein Inactive repressor

RNApolymerase

Promoter

trp operon

Genes of operon

Operator

mRNA 5

Start codon Stop codon

trpE trpD trpC trpB trpA

E D C B A

Polypeptide subunits that make upenzymes for tryptophan synthesis

(a) Tryptophan absent, repressor inactive, operon on

The trp Operon: Repressible Operons

•when tryptophan is absent – the operon needs to function to make tryptophan•the repressor protein is made but it is inactive & is incapable of binding the operator•RNA polymerase can bind the promoter and the downstream genes are expressed

(b) Tryptophan present, repressor active, operon off

DNA

mRNA

Protein

Tryptophan (corepressor)

Activerepressor

No RNAmade

The trp Operon: Repressible Operons

•when tryptophan is present – the operon does not need to be functional•tryptophan acts as a co-repressor & binds the repressor protein•this allows the repressor to bind and repress the function of the operator•MUCH lower downstream gene expression vs. when the operon is ON

• proposed by Francois Jacob and Jacques Monod - 1960s• E.coli can use glucose and other sugars (such as lactose) as their

sole source of carbon and energy• the normal situation is for the bacteria to use glucose

– levels of a bacterial enzyme called beta-galactosidase (lactose breakdown) are very low

• when lactose is given to the bacteria – b-Gal levels increase– said to be induced

• the lac operon is an inducible operon – contains genes that code for enzymes used in the hydrolysis and metabolism

of lactose• when E. coli are grown with glucose – no need for the enzymes of

the lac operon since there is no lactose in the medium– so the operon is turned OFF

• but with media containing lactose – need to turn the operon ON to make the enzymes for metabolizing and using lactose

The lac Operon: Inducible Operons

• genes of the lac-operon:– 1. lacZ gene = beta-galactosidase – splits the lactose into glucose and

galactose– 2. lacY gene – 3. lacA gene – 4. lacI gene = codes for a lac repressor– 5. operator = binds transcription factors– 6. promoter = binds RNA polymerase II

• THIS IS IMPORTANT!!! - without any outside control - the lac repressor gene lacI is constitutively active and acts to eventually switch the lac operon OFF– through the constitutive production of a lac repressor protein

• a molecule called an inducer is needed to inactivate the repressor to turn the lac operon ON

The lac Operon: Inducible Operons

(a) Lactose absent, repressor active, operon off

Regulatorygene

Promoter

Operator

DNAlacZlacI

DNA

mRNA5

3

NoRNAmade

RNApolymerase

ActiverepressorProtein

•when lactose is absent – an active repressor is made•the genes metabolizing lactose are NOT needed•repressor gene lacI is constitutively active – makes a lactose repressor•repressor binds the operator and hinders the binding of the RNA polymerase to the promoter•downstream genes are transcribed AT A VERY LOW LEVEL

lac repressor protein

(b) Lactose present, repressor inactive, operon on

lacI

lac operon

lacZ lacY lacADNA

mRNA5

3

Protein

mRNA 5

Inactiverepressor

RNA polymerase

Allolactose(inducer)

-Galactosidase Permease Transacetylase

•when lactose is present – an inducer is required to turn the operon ON•metabolizing and using lactose is now needed

•ALLOLACTOSE ACTS AS AN INDUCER•allolactose – form of lactose that can enter bacterial cells

•the inducer binds the repressor and prevents it from binding to the operator•the downstream genes are expressed AT A HIGH LEVEL

•lactose binding to the repressor shifts the repressor to its non-DNA binding conformation

• in nature – the inducer of the lab operon is a lactose derivative

• in the lab – other inducers can be used to turn the operon on– e.g. IPTG = isopropyl-b-D-thiogalactoside– IPTG is NOT a substrate of -Gal

• we can also give the bacteria a specific b-Gal substrate that will turn colors– X-Gal – turns blue with broken down by b-gal

enzyme– used to identify bacteria containing cloned genes– can insert additional genes into plasmids containing

the b-gal gene• insertion of your desired gene INTO the plasmid

disrupts -gal expression• inability to breakdown X-Gal – colonies are white

• inducible enzymes usually function in catabolic pathways– their synthesis is induced by a chemical signal

• repressible enzymes usually function in anabolic pathways– their synthesis is repressed by high levels of the end

product

• regulation of the trp and lac operons involves negative control of genes because operons are switched off by the active form of the repressor

Positive Gene Regulation: CAP proteins

• some operons are also subject to positive control• when bacteria are given both lactose AND glucose - the

bacteria will use glucose – the enzymes for glycolysis are continually present in bacteria

• when lactose is present and glucose is short supply – it makes the enzymes for lactose metabolism

• how does the bacteria sense the low levels of glucose??

Promoter

DNA

CAP-binding site

lacZlacI

RNApolymerasebinds andtranscribes

Operator

cAMPActiveCAP

InactiveCAP

Allolactose

Inactive lacrepressor

(a) Lactose present, glucose scarce (cAMP level high):abundant lac mRNA synthesized

-when glucose is scarce accumulation of a small molecule called cyclic AMP (cAMP)

-cAMP functions as a “2nd messanger” to signal that glucose levels are low in the growth medium- high levels of cAMP activate a regulatory protein called catabolite activator protein (CAP)-cAMP binds CAP and activates it- activated CAP attaches to the lac operon promoter and accelerates transcription (functions as a transcription factor)- enhances the affinity of RNA polymerase for the promoter

• CAP helps regulate other operons that encode enzymes used in catabolic pathways

• when glucose levels are low and lactose levels are high – 1. lactose binds the lactose repressor and prevents it

from binding the operator and inhibiting gene transcription = genes for lactose metabolism are made

– 2. cAMP activation of CAP and its binding to the lac promoter increases transcription = lactose genes are made at a higher rate

• when glucose levels increase and lactose levels decrease– 1. CAP activation will eventually decrease and so will its

enhancement of transcription– 2. the lactose repressor is now able to bind the

operator and inhibit transcription

• so the lac operon is actually under dual control as lactose increases and glucose decreases:– positive – as levels of cAMP rise – so does CAP activation and the activity of

the lac operon– negative – as repressor activity decreases & the activity of the lac operon

increases– THEREFORE: it is the allosteric state of the lac repressor that determines if

transcription happens– it is the presence of CAP that controls the rate at which transcription will

happen

Eukaryotic gene expression is regulated at many stages

• all organisms must regulate which genes are expressed at any given time

• in the same organism – the genomes are identical from cell to cell

• so why do different cells express different genes/proteins??

• differences result from differential gene expression = the expression of different genes by cells with the same genome

• several steps along the replication/transcription/translation path are control points for differential gene expression

– control of DNA transcription – modification of DNA-histone interaction

– post-transcriptional control– post-translational control

Signal

NUCLEUSChromatin

Chromatin modification:DNA unpacking involvinghistone acetylation and

DNA demethylationDNA

Gene

Gene availablefor transcription

RNA ExonPrimary transcript

Transcription

IntronRNA processing

Cap

TailmRNA in nucleus

Transport to cytoplasm

CYTOPLASM

mRNA in cytoplasm

TranslationDegradationof mRNA

Polypeptide

Protein processing, suchas cleavage and

chemical modification

Active proteinDegradation

of proteinTransport to cellular

destination

Cellular function (suchas enzymatic activity,structural support)

Control of DNA Transcription: Histone Acetylation

Histones

Histones

• each of the histone proteins (H2A, H2B, H3, H4) contain flexible extensions of 20 to 40 amino acids called “tails”

• these histones can be modified post-translationally by the addition of functional groups

• at the end of these tails are several positively charged lysine amino acids

• some of these lysines undergo reversible chemical modification called acetylation– important for transcription, resistance against

DNA degradation

Acetylation and Deacetylation of DNA• numerous post-translational modifications can be done to histone proteins

– affects how the DNA-histone interacts and ultimately affects the transcription of the DNA

• some histone lysines undergo reversible chemical modifications called acetylation and deacteylation

• acetylation = transfer of an acetyl group onto the NH2 terminus of an amino acid– for histones – performed by a family of enzymes called histone acteyltransferases

(HATs)• acetylation neutralizes the +ve charge of these lysines

– its interaction with the DNA is eliminated– the DNA becomes less tightly associated with the histone– results in better access for the transcriptional machinery to the DNA

lysineR-group

acetylcoA “donor”

Acetylation and Deacetylation of DNA

• deacetylation = removal of this acetyl group from the histone by a family of enzymes called histone deacetylases (HDACs)– increases the interaction between DNA and the histone by

removing the acetyl group and increasing the “positivity” of the lysine residues

– 11 eukaryotic HDACs !!!

lysineR-group

acetylcoA “donor”

Control of DNA Transcription: Acetylation and Deactylation of DNA

• acetylation/deacetylation is a transient histone modification that affects transcription– euchromatin – higher HAT activity more transcriptionally active form of

chromatin– heterochromatin – higher HDAC activity less transcriptionally active form

of chromatin

heterochromatin

euchromatin Protein

Increased bindingof transcription factorsand RNA Pol IIto “opened” acetylatedchromatin

Control of DNA Transcription: Acetylation and Deacetylation of DNA

• the HAT/HDAC enzymes are part of a large complex of proteins that binds the DNA

– includes transcription factors, other regulatory proteins, RNA polymerase II• it is now thought that HAT and HDAC enzymes are recruited into this complex

– once there – they modify the DNA and give the rest of the transcription machine better “access” to the DNA helix

• non-histone proteins can also be acetylated!!– e.g. transcription factors are also acetylated/deacetylated – changes their activity

level and therefore transcription

Histone Methylation

• histone methylation = the addition of methyl groups (CH3) to certain amino acids on histone tails– lysines or arginines – usually lysines– is associated with reduced transcription in cases, increased transcription in

others – usually results in increased association between the histone and the DNA

and a decrease in transcription in that area – histone methylation is considered an epigenetic modification

• alteration of gene expression by mechanisms outside of DNA structure• performed by a family of enzymes called histone methyltransferases

DNA Methylation• in addition to histones – methyl groups can be attached to

certain DNA bases = DNA methylation – usually cytosine– done by a different set of enzymes than those that methylate histones– is associated with reduced transcription in some species– i.e. the more methylated, the more inactive the gene

• DNA methylation essential for long-term inactivation of genes during cellular differentiation– DNA methylation can last through several rounds of replication– when a methylated DNA sequence is replicated – the daughter strand is

methylated too– can affect transcription rates over several rounds of replication

Regulation of Transcription Initiation

• chromatin-modifying enzymes provide initial control of gene expression by making a region of DNA either more or less able to bind the transcription machinery

• additional transcriptional levels are also found– enhancers– promoters

Enhancer(distal control

elements)DNA

UpstreamPromoter

Proximalcontrol

elementsTranscription

start siteExon Intron Exon ExonIntron

Poly-Asignal

sequence

Transcriptiontermination

region

Downstream

Organization of a Typical Eukaryotic Gene

• most eukaryotic genes are associated with multiple control elements– segments of noncoding DNA that serve as binding sites for transcription

factors that help regulate transcription– distal elements– known as enhancers– proximal elements – associated with promoters

• many of these control elements and the transcription factors they bind are responsible for the differential gene expression seen in different cell types

Transcription Factors• proteins that bind sequences of DNA to control transcription• can act as activators or repressors to transcription

– activating TFs - proteins that recruit the RNA polymerase to a promoter region– repressing TFs – proteins that prevent transcription in many ways

• must contain a DNA binding domain to be a transcription factor• not always one protein – can be multiple subunits together in a complex• two broad categories:

– 1. general transcription factors – 2. specific transcription factors

Transcription Factors• two broad categories:

– 1. general transcription factors are essential for the transcription of all protein-coding genes

• assist the RNA polymerase in binding the promoter region – only give a low level of transcription!!

• activity is enhanced by specific transcription factors– 2. specific transcription factors control the high-level, differential expression of specific

genes within a specific cell type• bind the promoter and enhancer regions of a gene• can function to activate or repress transcription• e.g. Runx-2 – transcription factor that is found in osteoblasts• directs the expression of several osteogenic genes involved in making bone

Transcription Factors bind DNA• binding of a TF to DNA is easy to see experimentally using a DNA mobility shift

assay• Step #1 – radioactively label your DNA• Step #2 – mix your labelled DNA with your possible TFs• Step #3 – Southern blot - run the DNA on a gel and transfer it to a filter paper

-control – DNA not mixed with the possible TF

• Step #4 – expose the filter to film to develop the radioactive signal

DNA footprinting assay

• sequence of DNA located immediately upstream of the transcription start site• promotes transcription of DNA into RNA• site of RNA polymerase binding in both prokaryotes and eukaryotes• contain sequences for the binding of RNA polymerase and sequences for the

binding of transcription factors

Promoters

• initial work done in bacteria– found two kinds of DNA sequences in the promoter

• 1. those that are found in the promoters of all bacterial genes• 2. those that are found in a more limited number of genes that respond to a

specific signal• core bacterial promoter: binds RNA polymerase and an associated sigma factor

(part of the RNA polymerase complex)– two key DNA sequences located 10 and 35 bp upstream from the transcription start site

(TSS) – for the binding of the RNA polymerase– -10 site = TATA box (consensus sequence TATAAT)– -35 site - TTGACA

Promoters

• eukaryotic promoters: 10 classes of promoters known– DEFINITION: DNA sequence that binds the RNA polymerase II (plus 7

additional factors)– BUT – more complicated than prokaryotic promoters– are additional upstream DNA sequences that regulate transcription– three well-known regions studied

• -30, -75 and -90 sites

– two kinds of DNA sequences associated with transcription of a eukaryotic gene:

– 1. part of the promoter (i.e. control elements) involved in the basic process of transcription

– 2. control elements active in a particular tissue type or in response to a specific signal – regulated transcription

Promoters

• promoter sequences involved in basic transcription– TATA box – conserved from the bacterial TATA box

• A/T rich sequence - 30 base pairs upstream of start site• in most genes but not all – not found in housekeeping genes• together with the transcription start site – considered to be the core promoter• accurately positions the RNA polymerase at the start site • also binds general transcription factors• actually provides a very low level of transcription • needs upstream promoter elements (UPEs) to increase transcriptional control

– UPEs – also essential for transcription• increase the efficiency of transcription • e.g. SP1 sequence &/or CCAAT box• some genes can have an SP1 and CCAAT box – e.g. hsp70 gene• others may only have one – e.g. metallothionein gene

Promoters

• promoters sequences involved in regulated transcription– promoters also have control elements that are shared with a more limited

number of genes– interspersed among the UPEs– provide cell-type specific or signal-specific transcription– some well-known sequences are known as Response Elements– e.g. metallothionein gene – metal response elements (MREs)

• gene binds metals such as zinc – acts to decrease oxidative stress

– many hormones act through these response elements• e.g. estrogen

Histone 2A gene promoter

Metallothionein 1 gene promoter

– functions of promoter were discovered through• mutation or deletion of specific regions • putting these regions in front of a reporter gene is the reporter gene

transcribed?• e.g. insertion of the metallothionein gene promoter into a reporter plasmid

containing the firefly luciferase gene (reporter gene)• transcription of the luciferase gene will be driven by the inserted promoter• put in the entire promoter and measure luciferase activity• put in “pieces” of the promoter with specific regions deleted – measure luciferase

activity

Promoters

Activation of Metallothionein Gene Expression by Hypoxia Involves

Metal Response Elements and Metal Transcription Factor-1

Cancer Res March 15, 1999 vol. 59 no. 6

pp1315-1322

• distal control elements of a gene• DNA sequences that act to enhance eukaryotic transcription• can be found either:

– upstream of the gene– downstream of the gene– within the gene– even on a different chromosome!!!

• act to increase the activity of the promoter– DO NOT have promoter activity themselves

• some enhancers are active in all tissues and increase promoter activity constitutively• others are only expressed in specific cells• made up of several DNA sequences (sequence elements) that bind transcription

factors which interact together• many of these sequence elements are also found in the promoter

Enhancers

Enhancer(distal control

elements)DNA

Upstream Promoter

Proximalcontrol

elementsTranscription

start siteExon Intron Exon ExonIntron

Poly-Asignal

sequence

Transcriptiontermination

region

Downstream

• transcription factors than bind enhancers are called activators– positively acting transcription factors

• activators = proteins that bind to DNA sequences and stimulate/activate transcription of a gene

• activators have two domains– 1. DNA binding domain– 2. activation domain - site that activates transcription by helping to form the transcription

initiation complex

Enhancers & their Transcription Factors

DNA

Activationdomain

DNA-bindingdomain

• so the typical eukaryotic gene consists of up to 4 distinct control elements– 1. core promoter itself – upstream of the transcription start

site– 2. upstream promoter elements (UPEs) located close to the

promoter – required for efficient transcription in any cell– 3. elements that intersperse among the UPEs and activate

transcription of genes in specific tissues or in response to specific stimuli – regulated transcription elements

– 4. distant elements called enhancers

Eukaryotic gene elements: a summary

Transcription Initiation

• transcription can happen as long as the core promoter is present– but transcription rates will be very low

• so efficient transcription of eukaryotic genes requires the activity of the promoter, enhancers and a multitude of transcription factors together with the RNA polymerase II

• these components come together to form a transcription initiation complex

• stepwise assembly– 1. binding of three general transcription factors at the TATA box– TFIIA,

TFIID TFIIB – 2. recruitment and binding of the RNA polymerase II at the TF/TATA box

complex• in some organisms – polymerase is bound to these 3 TFs before

binding DNA first = RNA polymerase holoenzyme– 3. additional general TFs join – 4. binding of gene-specific TFs + interaction with enhancer/activators

Activators

DNA

EnhancerDistal controlelement

PromoterGene

TATA box

Generaltranscriptionfactors

DNA-bendingprotein

Group of mediator proteins

RNApolymerase II

RNApolymerase II

RNA synthesisTranscriptioninitiation complex

-activators bind to the DNA of the enhancer via their DNA-binding domains-the activators bind to regions called distal control elements

a DNA bending protein “bends” the distal enhancer region – bringing it close to the the promoter

general transcription factors, promoter-specific TFs, mediator proteins and RNA polymerase II form a transcription initiation complex with the enhancer and its activators

Transcription Initiationpromoter-specific

transcription factorsactivator-enhancer

Mediator protein = co-activator-multi-subunit protein complex found in the transcription initiation complex of all eukaryotes-31 proteins!!!!-functions as a bridge between transcription factors

• some transcription factors can also function as repressors or silencers– inhibiting expression of a particular gene by a variety of

methods– some repressors bind activators and prevent their binding to

enhancers– some bind the distal control elements in the enhancer directly– others bind proximal control elements or the promoter

Repressors

Controlelements

Enhancer Promoter

Albumin gene

Crystallingene

LIVER CELLNUCLEUS

Availableactivators

Albumin geneexpressed

Crystallin genenot expressed

(a) Liver cell

Cell-Type Specific Transcription

• both liver and lens cells have the same genome

• so why does a liver cell make albumin and a lens cell make crystallin?????

• it’s the transcription factors and control elements

• liver cell has a unique complement of transcription factors that activate albumin transcription

Controlelements

Enhancer Promoter

Albumin gene

Crystallingene

LENS CELLNUCLEUS

Availableactivators

Albumin genenot expressed

Crystallin geneexpressed

(b) Lens cell

• the lens cell has a different set of TFs that activates crystallin transcription

• these transcription factors may only be made within a lens or liver cell at a precise time in development or in response to an extracellular signal (e.g. growth factor or hormone) or even an environmental cue

Coordinately Controlled Genes in Eukaryotes

• unlike the genes of a prokaryotic operon, each co-expressed eukaryotic gene has a core promoter and several other control elements

• these genes can be scattered over different chromosomes, but each has the same combination of control elements as one another

• multiple copies of activators recognize these control elements on each gene and promote their simultaneous transcription

Mechanisms of Post-Transcriptional Regulation• transcription alone does not account for gene expression• regulatory mechanisms can operate at various stages after transcription• allow a cell to fine-tune gene expression rapidly in response to

environmental changes• post-transcriptional processing:

– 1. mRNA structure – cap and tail; UTRs– 2. mRNA splicing– 3. mRNA half life and degradation

Post-Transcriptional Regulation: mRNA structure

• pre-RNA processing to mRNA involves the addition of the 5’ methylated cap and 3’ poly-A tail

• cap is added shortly after transcription initiation – by a capping enzyme which is associated with the RNA polymerase II

– cap - 7-methylguanosine– function of the cap

• 1. protection against mRNA degradation• 2. export out into the cytoplasm• 3. binding of the small subunit for translation

Post-Transcriptional Regulation: mRNA structure

• cap is followed by the 5’UTR region (untranslated region)– found between the transcription start site and ends one nucleotide before

the ATG/start codon of the coding sequence– ontains elements for controlling gene expression and mRNA export– contains sequences that are involved in translation initiation– in bacteria – contains a sequence for docking of the ribosome = Shine

Delgarno Sequence

TSS

Post-Transcriptional Regulation: mRNA structure

• poly A tail – in animal cells, all mRNAs (except histone mRNAs) have polyA tails

– prevent degradation of the mRNA and induces export from the nucleus– two special mRNA sequences are needed – located in the 3’UTR

• 1. Poly Adenylation signal – AAUAAA• 2. Poly A site – downstream from the signal – area rich in Gs ands Us

– area where the mRNA is cut and the poly-A tail is added

-complex of proteins binds the poly-Adenylation signal-a Poly(A) polymerase is recruited – cleaves the mRNA at the poly A site and adds

hundreds of “A” nucleotides

Post-Transcriptional Regulation: mRNA structure

• 3’UTR – second of the two UTRs that flank a transcription unit’s coding sequence– length is important – longer the UTR, the lower level of gene expression – can be an area of variability – controls expression levels and localization of a protein within a cell– contains numerous regulatory regions for

• 1. poly –adenylation – contains the polyA signal and polyA site• 2. mRNA creation – silencer regions to repress transcription of mRNA• 3. mRNA stability – contain AU-rich elements (AREs) that increase the stability of the

mRNA• 4. mRNA export – contains sequences that attract nuclear export proteins • 5. translation efficiency – also affected by AREs

Post-transcriptional control: mRNA degradation

• the life span of mRNA molecules in the cytoplasm is a key to determining protein synthesis

• eukaryotic mRNA is more long lived than prokaryotic mRNA• numerous enzymes (RNases) can breakdown mRNA• the binding of small RNAs called microRNAs (miRNAs) to

the mRNA can target it for degredation

Post-transcriptional Regulation: Splicing• removal of introns and the joining of exons• performed in the nucleus by the spliceosome

– small nuclear RNAs – U1, U2, U4, U5 and U6– associated with protein subunits = snRNPs– snRNPs form the core of the spliceosome

• spliceosome recognizes conserved sequences at the start and end of an intron = splice sites

• introns are removed as a lariat structure – a 5’ G at the end of the intron is joined in an unusual phosphodiester bond (2’ to 5’) to the A at the end of the intron

– “A” nucleotide is called a branch point

Post-transcriptional Regulation: Splicing• the snRNPs are numbered based on their

“entrance” into the splicing reaction• 1. first is U1 – base pairs with the 5’ G at the

start of the intron• 2. next is U2 which binds the branch point A• 3. U4/U6 and U5 enter next – formation of the

completed spliceosome• 4. rearrangements among these snRNAs “loops

out” the intron and cuts the intron at the 5’ end

• 5. departure of U1 and U4 + joining of the 5’ end of the intron to the A (completes the lariat)

• 6. U6 snRNA cuts at the 3’ end of the intron and joins the two exon sequences

Splicing

• In alternative RNA splicing, different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns

Exons

DNA

Troponin T gene

PrimaryRNAtranscript

RNA splicing

ormRNA

1

1

1 1

2

2

2 2

3

3

3

4

4

4

5

5

5 5

Initiation of Translation• initiation of translation of selected mRNAs can be blocked by regulatory

proteins that bind to sequences or structures of the mRNA• alternatively, translation of all mRNAs in a cell may be regulated

simultaneously• for example, translation initiation factors are simultaneously activated in an

egg following fertilization

Protein Processing

• after translation - various types of protein processing, including folding, cleavage and the addition of chemical groups take place

• known as post-translational processing• numerous kinds of chemical additions

Post-translational modifications of Proteins• usually involves enzymes adding

– 1. hydrophobic groups (fatty acids) for membrane localization of protein– 2. cofactors to enhance enzymatic activity – 3. additional peptides or proteins – e.g. ubiquitin– 4. structural changes – disulfide bridges between cysteines– 5. proteolytic cleavage– 6. chemical groups

• numerous chemical groups can be added– a. phosphorylation – b. methylation– c. acetylation– d. glycosylation– e. iodination

• non enzymatic modifications – changes the chemistry of an amino acid without the use of an enzyme– e.g. chemical addition of biotin (vitamin B7) as a cofactor

Protein Folding: Chaperones

• protein function is completely dependent upon 3D structure• the information for folding is contained within the amino acid sequence of the

polypeptide chain– the hydrophobic residues are “buried” within the center of the folding protein –

spontaneous process– large numbers of interactions between the R groups of the AAs form

• ionic• van der waals – between hydrophobic groups• disulfide bridges

• folding can begin the minute the PP chain emerges from the ribosome• but most protein don’t• these proteins are met at the ribosome by a class of proteins called molecular

chaperones

Protein Folding: Chaperones• molecular chaperones

– best described class of chaperones – heat shock proteins or HSPs– numerous families – e.g. HSP40, HSP60, HSP70– work by interacting with exposed hydrophobic AAs – hydrophobic residues are

dangerous– distinct mechanisms for each chaperone– e.g. HSP60 forms a “barrel-like” structure that “isolates” folding proteins AFTER they

are made = known as chaperonin– bind to the hydrophobic residues and ensures correct folding

• some chaperones bind to misfolded proteins and sequester them until they are destroyed

Protein tobe degraded

Ubiquitin

Ubiquitinatedprotein

Proteasome

Protein enteringa proteasome

Proteasomeand ubiquitinto be recycled

Proteinfragments(peptides)

• if the protein is not folded properly – will have to be degraded• Proteasomes are giant protein complexes that bind protein molecules and degrade them• consists of a protein complex that forms a hollow cylinder • top and bottom are additional protein complexes that feed the abnormal protein into the core

– protein is unfolded as it is fed in

– exposed to proteases within the core

– keeps the protein in the core until the entire protein is cleaved into peptides

• signal to enter the proteosome is the chemical attachment of a poly-ubiquitin chain– ubiquitin is prepared by an enzyme (Ubiquitin activating enzyme)– attached to lysines by enzymes (Ubiquitin ligase)– done over and over poly-ubiquitin chain

Noncoding RNAs play multiple roles in controlling gene expression

• only a small fraction of DNA codes for proteins– 30,000 to 100,000 genes

• a fraction of the non-protein-coding DNA pieces are genes for RNAs such as rRNA and tRNA

• but most are transcribed into noncoding RNAs (ncRNAs)– e.g. miRNA– e.g. siRNA

• noncoding RNAs regulate gene expression at two points: mRNA translation and chromatin configuration

MicroRNAs

(a) Primary miRNA transcript

Hairpin

miRNA

miRNA

Hydrogenbond

Dicer

miRNA-proteincomplex

mRNA degraded Translation blocked

(b) Generation and function of miRNAs

5 3

• MicroRNAs (miRNAs) are small single-stranded RNA molecules that can bind to mRNA

• can degrade mRNA or block its translation• miRNAs are made by RNA polymerase II & are capped

and poly-adenylated• exported out to the cytoplasm• a protein called Dicer cleaves the primary miRNA into the

mature miRNA • one strand of miRNA associates with proteins RNA-

induced silencing complex (RISC)• then the RISC base pairs with it complementary mRNA

nucleotides – usually in the 3’UTR• if base pairing is extensive cleavage of mRNA

– happens in plant cells

• if base pairing is limited repression of translation– happens in animal cells

• through RNA interference (RNAi) viruses can target and destroy host mRNAs

• RNAi is done through the production of small interfering RNAs (siRNAs) – target and destroy mRNA just like miRNA

• siRNAs and miRNAs are similar but form from Dicer cutting up different RNA precursors

– miRNA made from single stranded RNA made by the cell

– siRNA made from double stranded RNA (shRNA) made by viruses

• siRNA is now used in the lab to target specific mRNAs

– introduce artificial shRNA into cells siRNA that will bind and degrade your desired piece of mRNA

Small Interfering RNAs

1. viral production of dsRNA (shRNA)2. complexing with DICER siRNA3. integration of siRNA with RISC4. binding to target mRNA5. DESTRUCTION of mRNA

The Evolutionary Significance of Small ncRNAs

• Small ncRNAs can regulate gene expression at multiple steps

• An increase in the number of miRNAs in a species may have allowed morphological complexity to increase over evolutionary time

• siRNAs may have evolved first, followed by miRNAs