Transcription In Eukaryotes Translation

Transcription in Eukaryotes&Translation

Comparison of eukaryotic and prokaryoticpromoter recognitionEukaryotes: general transcription factors (GTFs).TFI factors for RNAP I, TFII factors for RNAP IIand TFIII factors for RNAP IIIProkaryotes: s factors

In addition to the RNAP and GTFs, in vivotranscription also requires– Mediator complex– DNA-binding regulatory proteins– chromatin-modifying enzymes

RNA polymerase II core promoters aremade up of combinations of 4different sequence elementsEukaryotic core promoter ( 40 nt): the minimalset of sequence elements required foraccurate transcription initiation by the Pol IImachinery in vitro4

Pol II core promoter TFIIB recognition element (BRE)The TATA element/boxInitiator (Inr)The downstream promoter element (DPE)5

Regulatory sequencesThe sequence elements other than the corepromoter that are required to regulate thetranscription efficiencyThose increasing transcription: Promoter proximal elements Upstream activator sequences (UASs) EnhancersThose repressing elements: silencers, boundaryelements, insulators6

RNA Pol II forms a pre-initiationcomplex with GTFs at the promoterThe involved GTFIIs (general transcriptionfactor for Pol II)– TFIID TBP (TATA box binding protein) TAFs (TBP association factors)– TFIIA, B, F, E, H7

1. TBP in TFIID binds to the TATAbox2. TFIIA and TFIIB are recruitedwith TFIIB binding to the BRE3. RNA Pol II-TFIIF complex isthen recruited4. TFIIE and TFIIH then bindupstream of Pol II to form thepre-initiation complex5. Promoter melting usingenergy from ATP hydrolysis byTFIIH )6. Promoter escapes after thephosphorylation of the CTDtail8

Promoter escape Stimulated byphosphorylation of theCTD (C-terminal domain)tail of the RNAP II– CTD contains theheptapeptide repeat TyrSer-Pro-Thr-Ser-Pro-Ser– Phosphorylation of the CTD“tail” is conducted by anumber of specific kinasesincluding a subunit of TFIIH

TBP binds to and distorts DNA using ab sheet inserted into the minor groove10

Correspondence between exons andprotein domainsGeneDNAExon 1Exon 2IntronIntron Exon 3TranscriptionRNA processingTranslationDomain 3Domain 2Domain 1Polypeptide

Translation Initiation in mRNAsShine Dalgarno Sequence in k’s Consensus Sequence in EukaryotesNNNNNNA/GNNAUGGNNNNNNNNNN

Translation: the basic peptideAminoacidsPolypeptideRibosometRNA withamino acidattachedGlytRNAAnticodonA A AU G G U U U G G CCodons5 mRNA3

5 3 Amino acidattachment siteHydrogenbondsA3 AnticodonA GAnticodon(c) Symbol(b) Three-dimensional structure5

An aminoacyl-tRNA synthetase joins aspecific amino acid to a tRNA

The anatomy of a functioning ypeptideExit P ASmallsubunit5 mRNA3 (a) Computer model of functioning ribosome. This is a model of a bacterial ribosome, showing its overallshape. The eukaryotic ribosome is roughly similar. A ribosomal subunit is an aggregate of ribosomalRNA molecules and proteins.

P site (Peptidyl-tRNAbinding site)A site (AminoacyltRNA binding site)E site(Exit site)LargesubunitEmRNAbinding sitePASmallsubunit(b) Schematic model showing binding sites. A ribosome has an mRNA binding site and three tRNAbinding sites, known as the A, P, and E sites. This schematic ribosome will appear in later diagrams.

Amino endGrowing polypeptideNext amino acidto be added topolypeptide chaintRNA3 mRNA5 Codons(c) Schematic model with mRNA and tRNA. A tRNA fits into a binding site when its anticodon base-pairs withan mRNA codon. The P site holds the tRNA attached to the growing polypeptide. The A site holds the tRNAcarrying the next amino acid to be added to the polypeptide chain. Discharged tRNA leaves via the E site.

The initiation of translationLargeribosomalsubunitP site3 U A C 5 5 A U G 3 Initiator tRNAGTPGDPEAmRNA5 3 5 3 Start codonmRNA binding siteSmallribosomalsubunit1 A small ribosomal subunit binds to a molecule ofmRNA. In a prokaryotic cell, the mRNA binding siteon this subunit recognizes a specific nucleotidesequence on the mRNA just upstream of the startcodon. An initiator tRNA, with the anticodon UAC,base-pairs with the start codon, AUG. This tRNAcarries the amino acid methionine (Met).Translation initiation complex2 The arrival of a large ribosomal subunit completesthe initiation complex. Proteins called initiationfactors (not shown) are required to bring all thetranslation components together. GTP providesthe energy for the assembly. The initiator tRNA isin the P site; the A site is available to the tRNAbearing the next amino acid.

The elongation cycle of translation1 Codon recognition. The anticodonTRANSCRIPTIONAmino endof polypeptideDNAmRNARibosomeof an incoming aminoacyl tRNAbase-pairs with the complementarymRNA codon in the A site. Hydrolysisof GTP increases the accuracy andefficiency of this step.TRANSLATIONPolypeptideEmRNARibosome ready fornext aminoacyl tRNA3 P Asite site5 2GTP2 GDPEEPPAA2 Peptide bond formation. AnGDP3 Translocation. The ribosometranslocates the tRNA in the Asite to the P site. The empty tRNAin the P site is moved to the E site,where it is released. The mRNAmoves along with its bound tRNAs,bringing the next codon to betranslated into the A site.GTPEPArRNA molecule of the largeSubunit catalyzes the formationof a peptide bond between thenew amino acid in the A site andthe carboxyl end of the growingpolypeptide in the P site. This stepattaches the polypeptide to thetRNA in the A site.

The termination of translationReleasefactorFreepolypeptide5 3 3 3 5 5 Stop codon(UAG, UAA, or UGA)1 When a ribosome reaches a stopcodon on mRNA, the A site of theribosome accepts a protein calleda release factor instead of tRNA.2 The release factor hydrolyzesthe bond between the tRNA inthe P site and the last aminoacid of the polypeptide chain.The polypeptide is thus freedfrom the ribosome.3 The two ribosomal subunitsand the other components ofthe assembly dissociate.

esIncomingribosomalsubunitsStart ofmRNA(5 end)End ofmRNA(3 end)(a) An mRNA molecule is generally translated simultaneouslyby several ribosomes in clusters called polyribosomes.RibosomesmRNA0.1 µm(b) This micrograph shows a large polyribosome in a prokaryoticcell (TEM).

The signal mechanism for targetingproteins to the ER1 Polypeptidesynthesis beginson a freeribosome inthe cytosol.2 An SRP bindsto the signalpeptide, haltingsynthesismomentarily.3 The SRP binds to areceptor protein in the ERmembrane. This receptoris part of a protein complex(a translocation complex)that has a membrane poreand a signal-cleaving enzyme.4 The SRP leaves, andthe polypeptide resumesgrowing, meanwhiletranslocating across themembrane. (The signalpeptide stays attachedto the membrane.)5 The signalcleavingenzymecuts off thesignal peptide.6 The rest ofthe completedpolypeptide leavesthe ribosome andfolds into its ecognitionparticleSRP(SRP)receptorCYTOSOL embraneProtein

Coupled transcription and translationin bacteriaRNA n oftranscription0.25 mDNAPolyribosomePolypeptide(amino end)RibosomemRNA (5 end)

The molecular basis of sickle-celldisease: a point mutationWild-type hemoglobin DNA3 Mutant hemoglobin DNA5 CT3 5 TCmRNAATIn the DNA, themutant templatestrand has an A wherethe wild-type templatehas a T.mRNAGAA5 G3 UA5 3 Normal hemoglobinSickle-cell hemoglobinGluValThe mutant mRNA hasa U instead of an A inone codon.The mutant (sickle-cell)hemoglobin has a valine(Val) instead of a glutamicacid (Glu).

Base-pair substitutionWild typemRNAA U G A A G U U U G G C U A A5 Protein3 MetLysPheAmino endGlyStopCarboxyl endBase-pair substitutionNo effect on amino acid sequenceU instead of CA U G A A G U U U G G U U A AMetLysMissensePheGlyStopA instead of GA U G A A G U U U A G U U A AMetLysPheSerStopNonsenseU instead of AA U G U A G U U U G G C U A AMetStopTransitionPurine to PurineAndPyrimidine to pyrimidineTransversionPurine to pyrimidineORPyrimidine to Purine

Base-pair insertion or deletionWild typemRNA5 ProteinA U G A A G U U U G G C U A AMetLysGlyPhe3 StopAmino endCarboxyl endBase-pair insertion or deletionFrameshift causing immediate nonsenseExtra UA U G U A A G U U U G G C U AMetStopFrameshift causingextensive missenseU MissingA U G A A G U U G G C U A AMetLysLeuAlaInsertion or deletion of 3 nucleotides:no frameshift but extra or missing amino acidA A G MissingA U G U U U G G C U A AMetPheGlyStop

A summary of transcription andtranslation in a eukaryotic cellDNATRANSCRIPTION1 RNA is transcribedfrom a DNA template.3 5 RNAtranscriptRNApolymeraseExonRNA PROCESSING2 In eukaryotes, theRNA transcript (premRNA) is spliced andmodified to producemRNA, which movesfrom the nucleus to thecytoplasm.RNA NUCLEUSAminoacidtRNAFORMATION OFINITIATION COMPLEXCYTOPLASM3 After leaving theAMINO ACID ACTIVATION4Each amino acidattaches to its proper tRNAwith the help of a specificenzyme and ATP.nucleus, mRNA attachesto the ribosome.mRNAGrowingpolypeptideActivatedamino acidRibosomalsubunits5 TRANSLATIONA succession of tRNAsadd their amino acids tothe polypeptide chainAnticodonas the mRNA is movedthrough the ribosomeone codon at a time.(When completed, thepolypeptide is releasedfrom the ribosome.)5EAAAAUG GUU UA U GCodonRibosome

A summary of transcription and translation in a eukaryotic cell TRANSCRIPTION RNA is transcribed from a DNA template. DNA RNA polymerase RNA transcript RNA PROCESSING In eukaryotes, the RNA transcript (pre-mRNA) is spliced and modified to produce mRNA, which moves from the nucleus to the cyt