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Chapter 31 Transcription 1. The role of RNA in protein synthesis 2. RNA polymerase 3. Control of transcription in eukaryotes 4. Posttranscriptional processing

Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

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Page 1: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Chapter 31Transcription

1. The role of RNA in protein synthesis2. RNA polymerase3. Control of transcription in eukaryotes4. Posttranscriptional processing

Page 2: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Three major classes of RNA

All participate in protein synthesis:

• Ribosomal RNA, rRNA1. Transfer RNA, tRNA2. Messenger RNA, mRNA

All are synthesized from DNA by transcription

Page 3: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

HistoricallyDNA found in cell nucleus, but RNA found in

cytosol (1930), microscopy and cell fractionationConcentration of cytosolic RNA-Protein particles

correlate with protein synthesis - is site ofprotein synthesis - was later identified asRibosome

In eukaryotes, DNA is never in association withprotein synthesis (Ribosome)

Incorporation of radiolabelled amino acids occurs inassociation with RNA-Protein particles

Structure of DNA revealed possible copymechanism

Page 4: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Central Dogma

DNA

RNA

Protein

Page 5: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

1. The role of RNA in proteinsynthesis

Studied by following enzyme induction

Bacteria vary the synthesis of certain enzymes depending on environmental Conditions

Enzyme induction occurs as consequenceof mRNA synthesis

Page 6: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Enzyme induction

E. coli can synthesize ˜4300 polypeptides

But enormous variation in abundance of specific polypeptides:

Ribosomal protein: 10’000 copies/cellRegulatory protein: <10 copies/cell

Housekeeping enzymes, constitutiveAdaptive, inducible enzymes

Page 7: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Lactose-metabolizing enzymes areinducible

E. coli is initially unable to metabolize lactose, but starts to induce the corresponding enzymes:

lactose permease, for uptakeβ-galactosidase, for splitting lactose

Few copies -> >10% or proteins, 1000-foldTriggering substance: inducer, allolactoseor IPTG

Page 8: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:
Page 9: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

The induction kinetics of β-galactosidase in E. coli

Page 10: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

The E. coli lac operon

Structural genesZ, β-galactosidaseY, lactose permeaseA, thiogalactisodase transacetylase

Control siteP, promoterO, operator

Regulatory geneI, inducer

Page 11: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Bacteria can transmitgenes via conjugation

Conjugation allows for bacterial genetics

Constitutive mutation: lac operon inducedeven without inducer -> mutation in I,distinct but closely linked to structuralgenes

PaJaMo experiment, 1956, Arthur PardeeFrancois Jacob, Jacques Monod

Page 12: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Bacterial conjugation

Page 13: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

F– cell acquires an Ffactor from an F+ cell

Bacterial conjugation:Transfer of genetic materialBetween a donor cell, F+, and recipient, F-

Ability to conjugate/mate is encodedon a plasmid, F factor (fertility)F+ cell are covered with F piliAllows binding to F- cell surface

Formation of cytoplasmic bridge and transfer of genetic informationConverts F- to F+

Page 14: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Transfer of the bacterialchromosome from an Hfr cell

to an F– cell and itssubsequent recombinationwith the F– chromosome

F factor can spontaneously integrateinto the genome -> Hfr strain(high frequency of recombination)

-> transmission of genomic informationupon mating:

in fixed ordertime dependent (90min)

Merozygote, partially diploidRecombination and integration

Page 15: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

The PaJaMo experimentMate Hfr I+Z+ to F- I-Z- in absence of inducerMonitor β-gal activity over time

Induction after 1h, cessation upon 2h -> Z+ in I- cells leads toconstitutive induction, cessation upon transfer of I gene -> Igene codes for diffusible repressor of Z, lac repressorF- is resistant to T6 and streptomycin

Page 16: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Messenger RNA

Second type of constitutive mutation Oc, operatorconstitutive, maps between I and Z genesIn merozygote F’ Oc Z- / F O+ Z+, β-gal inducibleBut in Oc Z+ / F O+ Z- constitutive synth of β-gal

O Can control Z only when on same chromosome !!!-> cis acting controlI is trans acting factor

Proteins are synthesized in two stages:1. DNA is transcribed in mRNA2. mRNA is translated into protein

This model explains behavior of lac system

Page 17: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Messenger RNA

In the absence of inducer, I binds to O andrepresses synthesis of structural genes Z, Y, A

On binding inducer, repressor dissociates from O,permitting transcription and subsequent translation

Operator-Repressor-Inducer system represents amolecular switch

Oc is constitutive because repressor cannot bind,Cis-acting element

Coordination of all 3 protein by single polycistronicmRNA transcript, cistron

Page 18: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

mRNAs have their predictedproperties

Kinetic of enzyme induction -> mRNA has to berapidly synthesized and degraded, short half-life

rRNA turnover is slower, comprises 90% of cellularRNA

Page 19: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

The distribution, in a CsCl density gradient,of 32P-labeled RNA that had been synthesized

by E. coli after T4 phage infection

Page 20: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

The hybridization of 32P-labeledRNA produced by T2-infected E.

coli with 3H-labeled T2 DNA

Page 21: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

2. RNA Polymerase

RNA polymerase is responsible for the DNA-directedsynthesis of RNA (1960), dNTP and DNA-dep.

E. coli RNAP hplpenzyme, 459kD, αββ'ωσ subunitcomposition, sigma σ70 unit dissociates from coreonce RNA synthesis has been initiated

RNAP functions:1. Template binding2. RNA chain initiation3. Chain elongation4. Chain termination

Page 22: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Components of E. coli RNAPolymerase Holoenzyme

Page 23: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Electron micrograph of E. coli RNA polymerase(RNAP) holoenzyme attached to various promoter

sites on bacteriophage T7 DNA

Page 24: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Template bindingo RNA synthesis is initiated only at specific sites on theDNA templateo RNAP binds to its initiation sites at sequence elementscalled promoter, these are recognized by sigma factor (K≈10-14M)o Promoter, ca. 40bp element, located 5’ of structuralgene, first base in RNA is +1, initiation siteo If RNAP bound to promoter -20 to +20 are DNaseIprotectedo Consensus promoter sequence, hexamer centered at -10 = Pribnow Box, TATAAT, plus additional element at -35 sequence in between not important, but distanceo +1 is either A or G

Page 25: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

The sense (nontemplate) strandsequences of selected E. coli promoters

Rate of transcription varies 1000-fold,correlates with strength of promoter to bind RNAP

Page 26: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Initiation requires formation ofan open complex

RNAP binding alters accessibility of bases towardsmethylating agents (dimethyl sulfate, DMS), DMS-footprinting -> holoenzyme binds to only one side/face ofDNA and melts DNA

Page 27: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Chain initiation+1 is purine, A > GInitiation reaction: pppA + pppN -> pppApN + PpiUnlike DNA replication, RNA initiation does not need aprimer

Crab claw shape of Taq RNAP, pincers formed by β andβ ’ with a cavern between the two pincers

Prokaryotic initiation is inhibited by rifamycin B,Steroptomyces mediterranei, rifampicin commercial gram-positive antibiotic (also tubeculosis), does not blockpromoter bdg or elongation, but only initiation

Page 28: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Structure of Taq RNAP coreenzyme

Page 29: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Chain elongation5’ -> 3’ or 3’ -> 5’ growth ?

Label with γ[32P]GTP, chase with cold GTPIf 5’->3’, RNA is permanently labelledIf 3’->5’, RNA is unlabelled

Page 30: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Transcription supercoils DNAElongation requires opening of dsDNA, bubble2 models:

RNAP swirls around DANN -> transcript would wrapDNA rotates -> DNA must be tethered

Page 31: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Transcription occurs rapidly andaccurately

In vivo rate is 20-50nt/sec, entirely processive, no exo-nucleolytic correction like in DNA pol.One mistake per 10’000nt, tolerable because:

1. Genes are repeatedly transcribed2. Genetic code is redundant -> high prob. of silent mut.3. Aa substitutions in proteins are often tolerated4. Large portion of transcript is non-coding (intron)

Page 32: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Intercalating agents inhibitboth RNA and DNA polymerase

Actinomycin D intercalates into DNA andRNA, inhibits transcription and replication

Page 33: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Chain TerminationEM specific sites of termination, two common features in E.coli: 1. Series of 4-10 consecutive A-Ts, As on template

2. G + C-rich palindromic region upstream of A-Ts

Page 34: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

A hypothetical strong(efficient) E. coli terminator

The stability of terminator G+C hairpin + weak base pairingBetween RNA polyU and DNA template ensure termination

Page 35: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Termination often requires theassistance of Rho factor

50% of termination sites lack cis-acting terminator sequences, but require protein factor, Rho

in vivo transcripts often shorter than in vitroRho is hexameric, 419AaHelicase activityRecognition sequence on RNA transcript

Page 36: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Eukaryotic RNA polymerase3 distinct types of RNAP

1. RNAP I, in nucleoli, makes rRNA2. RNAP II, in nucleoplasm, makes mRNA precursors3. RNAP III, nucleoplasm, 5S rRNA, tRNA, small RNAs

Up to 600kD, up to 12 subunits, 5 of these present in all 3 RNAP types

RNAP II has extraordinary C-terminal domain, CTD52 repeats of PTSPSYS, 50 Ser are phosphorylatedTranscription is only initiated if CTD is unphosphorylatedElongation occurs only if CTD is phosphorylatedPhosph. Converts initiation complex to elong. compl.

Page 37: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

RNA Polymerase Subunitsa

Page 38: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

X-Ray structure of yeast RNAPII that lacks its Rpb4 and

Rpb7 subunits

Resembles Taq RNAPcrab claw like shape

Page 39: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Cutaway schematic diagram of thetranscribing RNAP II elongation

complex

On DNA binding, 50kD clamp swings out -> processivity

Page 40: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Amatoxins specifically inhibit RNApolymerase II and III

Poisonous mushroom, Amanitia phalloidesResponsible for majority of fatal mushroom poisoningsToxin, bicyclic octapeptide, amatoxins, α-amanitinTight 1:1 complex with RNAP, K 10-8MAct slowly, death after a few days -> turnover of RNA

Page 41: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Mammalian RNAP I has a bipartitepromoter

Numerous rRNA genes have essentially identical sequenceand promoterBut unlike RNAP II and RNAP III, RNAP I promoters areSpecies specific !!

Core promoter -31 to +6 and upstream element (-187 to -107)

Page 42: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

RNAP II promoters are complexand divers

Euk RNAP II promoters are more complex than theirprokaryotic homologues

GC-box upstream of constitutive genesSelectiveley expressed genes often contain TATA box (-27 to -10), resembles -10 of prok. Genes

Mutation sin TATA box cause heterogeneity in initiation

CCAAT box, -70 tp -90, i.e. in globin genes

Page 43: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

The promoter sequences of selectedeukaryotic structural genes

Page 44: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Enhancers are transcriptional activatorsthat can have variable positions and

orientationsPromoter element that act in both orientation and distanceindependent = enhancers

Can act from several kb, in euk. Viruses or structural genes

Required for full activity of promoter

Recognized by specific transcription factors -> DNA loopStimulate entry of RNAP II on promoter

Mediate much of selective gene expression

Page 45: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

RNAP III promoters canbe locateddownstream from their transcription

start siteRNAP III promoters can be within the gene’s transcribedregion !! 5S rRNA

Page 46: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

3. Control of Transcription inProkaryotes

Adaptation to environmentalchanges takes only minutesbecause transcription andtranslation in prokaryotes arecoupled (euk. takes hours)

mRNAs are degraded in 1-3min

Page 47: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Promoters

Genes that are transcribed at high rates have efficientpromoters

Lac I is transcribed at 10 copies/cell

Page 48: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Gene expression can be controlledby a succession of sigma factors

Cell development and differentiation involves thetemporally ordered expression of specific sets of genesaccording to a genetically specified program

For example phage infection in prokaryontes1. Expression of early genes2. Expression of middle / late genes

One way of regulation: cascade of sigma factors thatrecognize the respective promoters

Page 49: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

lac Repressor I: Binding

1966 isolation of repressor based on binding to radio-Labelled IPTG, protein low abundance (0.002%)

Tetramer, 360 Aa, binds DNA K = 10-6M, promoter 10-13MTrypsin cleavage releases two domains. N-term. binds DNArest binds IPTG

Protein scans DNA to bind to promoter (on rate is greaterthan diffusion limited process).

Page 50: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

lac operator has a nearlypalindromic sequence

Repressor protein used to “fish” binding DNA sequence

Lac I binds to 26bp element with nearly 2-fold symmetry

Page 51: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

lac repressor prevents RNA polymerase fromforming a productive initiation complex

RNA polymerase binds +20 to -20Operator occupies +28 to -7-> lac operator and promoter overlapBinding of repressor obstructs RNAP binding

Page 52: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Catabolite Repression: An exampleof gene activation

Glucose is the metabolite of choice !!!In its presence, no other C-source is being metabolized,>100 enzymes are repressed (arabinose, galactose, lactose)= catabolite repression

Prevents wasteful duplication of energy producingenzymes

Page 53: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

The kinetics of lac operon mRNA synthesisfollowing its induction with IPTG, and of its

degradation after glucose addition

Page 54: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

cAMP signals the lack of glucose

cAMP is second messenger in animal cellsIn E. coli, cAMP greatly diminished in presence of glucose

Addition of cAMP to culture overcomes cataboliterepression

Page 55: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

CAP-cAMP complex stimulates thetranscription of catabolite repressed operons

cAMP binding protein = catabolite gene activator protein,CAP = cAMP receptor protein, CRP

Homodimer of 210 Aa, undergoes large conformationalchange upon cAMP bdg.

CAP-cAMP binds lac operon and stimulates transcription ->positive regulator (unlike lac I, negative regulator)

Binds DNA and bends it 90°, contacts CTD of RNAP

Page 56: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

X-Ray structures of CAP–cAMPcomplexes

Page 57: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Sequence-specific protein-DNAinteractions

Genetic expression is controlled by proteins such as CAP, lac repressor

How do proteins bind to specific DNA sequences, how dothey recognize base (pairing) ?

Base position in minor (5Å wide, 8Å deep groove is sequence independent, but varies in major groove ! -> protein / base recognition via major groove, 12Å wide, 8Å deep

Page 58: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

The helix-turn-helix is a common DNArecognition element in prokaryotes

Cap dimer’s two symmetrical F helices fit into two successivemajor grooves of B-DNA

CAP’s E and F helix form a helix-turn-helix (HTH) motif(supersecondary structure), similar to lac repressor, trp repressor

HTH is 20 Aa motif, helices cross at 120°

F helix in CAP is recognition helix, complex structural interactions (hydrogen bonds, salt bridges and van der Waalsinteractions), i.e. no simple Aa - Base code

Page 59: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

HTH-DNA interaction

Page 60: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

araBAD operon:positive and negativecontrol by the same protein

Arabinose is not metabolized by human, but E. coli in our gutwill metabolize this pentose

5 enzymes form an catabolite repressible araBAD operon

Control sites araI, araO1, araO2

Regulator: araC, homodimer 292Aa, N-term arabinose bindingand dimrization domain, linker + C-term DNA bdg. domain

Page 61: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Genetic map of the E. coli araCand araBAD operons

Page 62: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Mechanism of araBAD regulation

Page 63: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

lac repressor II: structureDNA loop formation is an important mechanism for transcriptional regulation

The lac repressor is a dimer of dimers, V-shaped

Page 64: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Model of the 93-bp DNA loop formedwhen lac repressor binds to O1 and O3

DNA loop is furtherstabilized by cAMP-CAPbinding

Principal:Modular build up andbreak down of veryhigh affinity complexes

Page 65: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

trp operon: Attenuation

Attenuation: Control mechanism to regulate amino acid biosynthetic operons

E. coli trp operon: five polypeptides, 3 enzymes,mediate the synthesis of trp from chorismate

Regulated by trp repressor, homodimer, 107Aa, bind L-trp -> binds trpO -> repressionTrp acts as a corepressor

Page 66: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Genetic map of the trp operon

Page 67: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Tryptophan biosynthesis isregulated by attenuation

trpE, first structural gene in trp operon is preceded by trpL, 162nt leader sequence

Availability if trp results in premature transcription Termination within trpL

Control element for this transcription termination = attenuator

trpL contains 2 consecutive trp codons thereby couplesTranslation rate to formation of RNA secondary structureand transcription termination

Similar in his operon, ilv operon

Page 68: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

The alternative secondarystructures of trpL mRNA

Page 69: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

The trp attenuator’s transcriptiontermination is masked when trp is scarce

Page 70: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Amino Acid Sequences of SomeLeader Peptides in Operons Subject

to Attentuation

Page 71: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Regulation of rRNA synthesis:The stringent response

E. coli division 20 min, contains 70’000 ribosomes -> must synthesize 35’000 ribosomes/20 min

Initiation of rRNA transcription : 1 /sec -> 1200 ribosomes/20min

⇒Seven distinct rRNA operons per chromosome + multiple replicating chromosomes

Coordination: rate of rRNA synthesis proportional to rateof protein synthesis

Molecular control of this coordination: Stringent Response

Page 72: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

(p)ppGpp mediates the stringentresponse

o The stringent response is correlated with a rapid intracellularaccumulation of two unusual nucleotides:

ppGpp and pppGpp = (p)ppGpp

o Rapid decay when amino acids become available

o relA- mutants exhibit no stringent response = relaxed control lack(p)ppGpp

o (p)ppGpp inhibits the transcription of rRNA genes, butstimulates transcription of trp and lac operons

o (p)ppGpp alters RNAP promoter specificity

o Rel A, stringent factor: ATP + GTP <-> AMP + pppGppo Active in association with ribosome engaged in translation but lackcharged tRNAso (p)ppGpp degradation by SpoT

Page 73: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

4. Posttranscriptional Processing

Primary transcripts - of eukaryotes - are not yetfunctional but undergo post-transcriptionalmodifications:

1. Exo- and endonucleolytic removal of nt2. Appending nt at 3’ and 5’ ends3. Modification of specific nucleosides

Page 74: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Messenger RNA processing:caps, tails, and splicing

In eukaryotes: primary transcripts are made in thecell nucleus, but translation takes place in the cytosol

Primary transcripts are processes on their transport wayto the cytosol

Page 75: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Eukaryotic mRNAs are capped

Cap: 7-methylguoanosine is joinedat 5’ nucleoside via5’-5’ triphosphate bridge

Cap defines eukaryotic translationstart site

Addition requires:1. RNA triphosphatase2. Capping enzyme3. Guanine-7-methyltransf.4. 2’-O-methyltransf.

Page 76: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Eukaryotic mRNAs have poly(A) tailsUnlike prokaryotic mRNAs, eukaryotic mRNAs arealways monocistronic.

Termination process in imprecise -> heterologous 3’ends, but mature mRNAs have well defined 3’endswith tails of ~250 polyAdenosine nucleotides

Appended by two reactions:1. Cleavage of (heterologous) 3’ end, ~20nt

past AAUAAA sequence; CFI, CFII2. Poly(A) polymerase, PAP

Poly(A) tail gets shorter as mRNA agesMature histone mRNAs lack poly(A) tail

Page 77: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Eukaryotic genes consist of alternatingexpressed and unexpressed sequences

Primary transcripts are heterogenous in size and muchlarger than mature mRNAs !! ->heterogenous nuclearRNA (hnRNA) mature to mRNAs by excision of internalsequences (pre-mRNA)Intervening sequence = intron (~1500nt, average 8/gene)Expressed sequence = exon (~300nt)Largest gene, titin 29’926 Aa, 234 introns, 17kb exon

Page 78: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Exons are spliced in a two-stagereaction

Gene splicing must be precise to maintain the readingframe !!

Page 79: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

The consensus sequence at theexon–intron junctions of vertebrate pre-

mRNAs

Invariant GU at intron 5’ boundary, AG at 3’ boundary

Page 80: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

The sequence of transesterification reactionsthat splice together the exons of eukaryotic

pre-mRNAs

Page 81: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Types of Introns

Page 82: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Exons are spliced in a two-stagereaction (2)

1. Formation of 2’,5’-phosphodiester bond betweenadenosine in intron and 5’ phosphate-> intron assumes lariat structure

2. Free 3’-OH of 5’ exon generates phosphodiesterwith 3’ exon, -> releasing the intron lariat

Intron lariat is then debranched, and degraded

Note. No free energy input !

Page 83: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Some eukaryotic genesare self-spliced

Today we know 8 distinct types ofintrons:

Group I introns, nuclei,mitochondria, chloroplastsTetrahymena, ciliate, no proteinrequired for splicing, RNA only+ guanosine

Self-splicing RNA = ribozyme

Page 84: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Group II intronsMitochondria of fungi and plantsSelf splicing, lariat intermediate but no externalnucleotide

Spliceosome is an RNA-protein complex that mediatessplicing of normal pre-mRNA,evolved from group primordial self-splicing RNA,Protein thought to be important for fine-tuning ofribozyme structure,

Similar, RNA of ribosome has catalytic activity

=> RNA world hypothesis

Page 85: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

The self-splicing group I intronfrom Tetrahymena thermophila

Page 86: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Hammerhead ribozymes catalyze anin-line nucleophilic attack

Simplest and best characterized ribozymesEmbedded in the RNA of certain plant virusesTermed hammerhead enzyme due to structural resemblance

Enzyme greenSubstrate blueCleavage site red

Page 87: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Splicing of pre-mRNAs is mediatedby snRNPs in the spliceosome

o How are splicing junctions recognized and how are thetwo exons joined ?

o Eukaryotes contain many 60-300nt nuclear RNAs,termed small nuclear RNAs, snRNA

o Form RNA-protein complexes termeds small nuclearribonucleoproteins, snRNPs

o U1-snRNA (U-rich) is complementary to 5’ splice site,recognizes this splice site

o Splicing takes place in 45S particle, spliceosome, whichbrings together pre-mRNA and snRNPs, 5 RNAs, ~65proteins, ATP-dep., U2-,U4-, U5-, U6-snRNPs

Page 88: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Figure 31-55 The X-raystructure of the catalyticpocket in the hammerhead

ribozyme’s kinetically trappedintermediate.

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Page 89: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

An electronmicrograph ofspliceosomes in

action

Page 90: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

A schematic diagram of six rearrangements that thespliceosome undergoes in mediating the first

transesterification reaction in pre-mRNA splicing

1. Exchange of U1 for U6 in base pairing to 5’ splice site2. Exchange of BBP for U2 in binding to branche site3. Intramolecular rearrangement in U24. Disruption of pairing between U4 and U65. Disruption of a second stem between U4 and U66. Disruption of a stem-loop in U2

Page 91: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Splicing also requires theparticipation of splicing factors

o Variety of proteins known as splicing factors that arenot part of the spliceosome also participate in the splicingreaction

o Branche point binding protein, BBP (=SF1, U2AF)

o SR proteins (Ser, Arg-rich) and members of theheterogenous nuclear ribonucleoprotein family (hnRNP),contain RRM domain (RNA Recognition Motif), hnRNP arehighly abundant

o Exon skipping does not normally occur, splicing occursorderd in 5’->3’ direction, cotranscriptional

Page 92: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Structure of the RNA binding portion ofhuman branch point-binding protein (BBP) in

complex with its target RNA

Page 93: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Spliceosomal structureso All 4 snRNPs involved in pre-mRNA splicing contain the samesnRNP core protein, which consist of 7 Sm proteins (reactwith autoantibodies from patients with systemicerythematosis), named B/B’, D1, D2, D3, E. F, and G protein

o Each of these Sm proteins contain two conserved segments,Sm1 and Sm2 separated by a variable linker

o The seven Sm proteins bind to conserved RNA sequence,the SM RNA motif, occurs in U1-, U2-, U4, and U5-snRNA

o Form heptameric ring, central hole positive charged allowspassage of ss RNA but not ds RNA

o U1-snRNP consist of 10 proteins, 7 Sm proteins and 3 U1specific factors

Page 94: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

A model of the snRNP coreprotein

Page 95: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

The electron microscopy-based structure ofU1-snRNP at 10 Å resolution

The predicted secondary structure of U1-snRNA The molecular outline of U1-snRNP

Page 96: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Significance of gene splicing

o Why are there introns ?o Introns are rare in prokaryotic structural geneso Uncommon in yeast, 239 introns in 6000 geneso Abundant in higher eukaryoteso Histones lack intronso Unexpressed sequences constitute 80% for a typical

vertebrate structural geneo Molecular parasites (junk DNA) ?o Evolution of complex spliceosome must have been

advantageous over elimination of split genes

o intron/exon organization is:- An advantage for rapid evolution of new proteins- Allows gene function tuning through alternative splicing

Page 97: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Many eukaryotic proteins consist ofmodules that also occur in other proteins

o Example, LDL-receptor839 Aa, 45kb gene, 18 exons, most encode specific

functional domains, 13 of these segments/domains havehomology with domains found in other proteins=> modular construction of the LDL receptor=> modular construction is found in many other proteinsthat are composed of re-utilized domains (i.e. signaltransduction, SH2, SH3 domains etc.)

Page 98: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Alternative splicing greatly increases the numberof proteins encoded by eukaryotic genome

o The expression of numerous cellular genes is modulated bythe selection of alternative splice sites

o Example rat α-tropomyosin gene encodes 7 tissue specificvariants of the muscle protein

Page 99: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Alternative splicingo Occurs in all metazoanso Human genome only 30’000 genes but estimated 50’000 -

140’000 structural genes

o Entire functional domains or single amino acids can bealtered in proteins through alternative splicing

- soluble or membrane bound- can be phosphorylated by a specific kinase or not- subcellular localization- whether enzyme binds a specific allosteric effector- affinity of receptor - ligand interaction

o Selection of alternative splice sites is developmental andtissue specific (regulation in space and time)

Page 100: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Selection of alternative splice sitesBest understood for Drosophila sex-determination genes

no TRA protein functional TRA protein(repression of splice siteby Sxl)

Male-specific DSX protein -> represses female-specific genesFemale-specific DSX protein -> represses male-specific genes(activation fo splice site)

Page 101: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

AU-AC introns are excised by a novelspliceosome

o Small fraction of introns (~0.3%) have AU rather than GUat their 5’ends and AC rather than AG at 3’But are excised via lariat intermediateAre splice by a AU-AC spliceosome with U5 sn RNP incommon but specific U11, U12 and U4atac-U6atac

Page 102: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Trans-splicing

o Trans-splicing, joining of two separate RNA molecules- Observed in Trypanosomas, all mRNAs have same 35nt

leader, but this leader is not present in thecorresponding genes

- Splice leader (SL) RNA, transcribed from anindependent gene

- Trans-splicing reaction resembles spliceosome-mediatedcis-splicing

- But Y-shaped rather than lariat intermediate

Page 103: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

The sequence of transesterificationreactions that occurs in trans-splicing

Page 104: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

RNA can be edited by the insertion ordeletion of specific nucleotides

o Certain RNA differ in sequence from their correspondinggenes

Examples: C->U and U->C changesInsertion or deletions of UInsertion of multiple G or C residues

o Most extreme case in mitochondria of Trypanosomesinvolves addition and removal of hundreds of U’s to andfrom 12 otherwise untranslatable mRNAs

o RNA editing, violates central dogma ? Because nottemplate-based

o Discovery of guide RNAs (gRNAs), 50-70nt, 3’ oligo(U) tail

Page 105: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

A schematic diagram indicating how gRNAsdirect the editing of trypanosomal pre-edited

mRNAs

RNA editing occurs on ~20S RNP, editosomegRNA is used as template to “correct” the mRNA Requires: 1. Endonuclease

2. Terminal uridylyltransferase3. RNA ligase

Page 106: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Trypanosomal RNA editingpathways

Page 107: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

RNA can be edited by base deamination

o Humans express two forms of apolipoprotein B (apoB):- apoB-48, only made in intestinal tissue, functions inchylomicron transport, triacylglycerol to liver and periphery- apoB-100, made in liver, functions in VLDL, IDL, and LDLto transport cholesterol from liver to periphery

o apoB-100 is 4536Aa large protein, apoB-48 consists ofapoB-100 N-terminal 2152 residues but lacks C-term domainof apoB-100 that mediate LDL receptor binding

o Both are expressed from the same gene, mRNAs differ in asingle C->U change, resulting in stop codon (UAA)

o Base change mediated by a protein, cytidine deaminasesubstitutional editing, also in glutamate receptor

Page 108: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

RNA interferenceo Noncoding RNAs can have important roles in controlling

gene expressiono Anti-sense RNA can block translation of a specific message Yet injection of sense RNA into C. elegans also blocks

protein productiono Added RNA interferes with gene expression = RNA

interference, RNAio 1998 Andrew Fire and Craig Mello, Nobelprice 2006 ds RNA is substantially more efficient in causing RNAi

induced by only a few molecules -> catalytic rather thanstoichometric

Page 109: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

A model for RNA interference (RNAi)

1. Trigger RNA is cut to 21-23ntoligos =small interf. RNAsiRNA, with 2nt overhang at 3’ and5’ phosphatemediated by RNase,Dicer

2. siRNA is transfered tomultisubunit RISC complex, RNA-induced silencing, siRNA guidessubstrate specificity

3. RISC cleaves mRNA, which is thenfurther degraded

RNAi requires that trigger dsRNAis copied, mediated by RNA-dependent RNA polymerase (RdRP)

Method of choice for knockoutstudy

Page 110: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

A model for transitiveRNAi

siRNA can prime for RdRP-catalyzed synthesis of secondarytrigger dsRNAs which are dicedto yield secondary siRNAs

May yield non-specific silencing,Transitive RNAi

RNAi may arose as defenseagainst RNA viruses, inhibitmovement of retrotransposons

Page 111: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Ribosomal RNA Processing

o Seven rRNA copies in E. coli genome,

o polycistronic >5500nt transcript, containing 16S rRNA, 1-2tRNAs, 23S rRNA, 5S rRNA, plus 1-2 more tRNAs at 3’

o Processing into mature rRNAs, cotranscriptional

o Specific endonucleolytic cleavage by RNase III, RNase P,RNase E, RNase F

o Secondary processing, trimming of 5’ and 3’ ends occurswhile rRNA is already associated with ribosomal proteins

Page 112: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

The posttranscriptionalprocessing of E. coli rRNA

Page 113: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Ribosomal RNAs are methylated

o During ribosome assembly, 16S and 23S rRNA is methylatedat 24 specific nucleosides, SAM-dep.

o N6,N6-dimethyladenine and O2-methylribose (protect fromRNase degradation)

Page 114: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Eukaryotic rRNA processing is guidedby snoRNAs

o rRNA transcription and processing takes place in nucleolio Primary transcript is ~7500nt 45S RNA that contains 18S,

5.8S, 28S rRNAs separated by spacer sequenceso Specific methylation (106 sites in humans)o Conversion of U to pseudouridine (95 in humans)o Subsequent cleavage and trimming analogous to prok.

o How are methylation sites recognized/targeted ?o Pre-rRNA interact with small nucleolar RNAs, snoRNAs

( ~200 in mammals), intron-encoded

Page 115: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

The organization of the 45S primarytranscript of eukaryotic rRNA

Page 116: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Transfer RNA processing

o tRNA, ~80nt, cloverleaf secondary structure, large fractionof modified bases

o E. coli chromosome contains 60 tRNA genes, some arecomponents of rRNA operons

o Primary transcript contains 1-5 tRNA copies, excision andtrimming similar to rRNA processing

o Many tRNAs contain intronso CCA trinucleotide to which the amino acid is appended is

postracriptionally added, tRNA nucleotidyltransferase

o RNase P generates 5’ ends of tRNAs, contains 377nt RNAcomponent, is catalytic subunit

Page 117: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

A schematic diagram of the tRNAcloverleaf secondary structure

Page 118: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Figure 31-74a The structureof the RNA of B. subtilisRNase P. (a) Predicted

secondary structure withspecificity domain drawn invarious colors and catalytic

domain is black.

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Page 119: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

Figure 31-74b The structureof the RNA of B. subtilisRNase P. (b) The X-ray

structure of the specificitydomain in which its various

segments are colored as in Parta.

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Page 120: Voet Chapter 31 - unifr.ch · 2008. 9. 6. · PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod. ... I is trans acting factor Proteins are synthesized in two stages:

The posttranscriptional processingof yeast tRNATyr