Biol 434 1st Edition Exam # 2 Study Guide Lectures: 1 – 12TRANSCRIPTION IN EUKARYOTESIntroduction-There are differences in the machinery between prokaryotes and eukaryotes: bacteria only have one RNA polymerase whereas all eukaryotes have at least three (Pol I, II, III)-Bacteria only require one additional initiation factor (σ); several factors are required for efficient and promoter-specific initiation in eukaryotes. These are called general transcription factors (GTFs)-In vitro, the GTFs are all that are required, together with Pol II, to initial transcription on a DNA template (no histones).-In vivo, however, the DNA template in eukaryotic cells is incorporated into nucleosomes. Under these circumstances, the GTFs are not alone sufficient to bind promoter sequences and elicit significant expression. Rather, additional factors are required, including DNA-binding regulatory proteins, the so-called Mediator complex, and often chromatin-modifying enzymes.RNA Polymerase II core promoters are made up of combinations of different classes of sequence element-The eukaryotic core promoter refers to the minimal set of sequence elements required for accurate transcription by the Pol II machinery, as measured in vitro. A core promoter is typically 40-60 nucleotides long, extending either upstream/downstream from start site.RNA Pol II forms a preinitiation complex with general transcription factors at the promoter-The GTF collectively perform the functions of the σ in bacterial transcription.-It helps polymerase bind to the promoter and melt the DNA (similar to closed to open transition)-They also help polymerase escape and embark on the elongation phase.-The complete set of GTFs and polymerase, bound at promoter, and poised for initiation is the preinitiation complex.Promoter Escape Requires Phosphorylation of the Polymerase "Tail"-Abortive initiation before elongation-In prokaryotes: polymerase synthesizes a series of short transcripts.-In eukaryotes, promoter escape involves two steps not seen in bacteria: one is ATP hydrolysis (in addition to the one for DNA melting), and the other is phosphorylation of the polymerase.TBP Binds to and distorts DNA Using beta sheets inserted into the minor groove-Beta sheets recognize minor groove of TATA element.The other general transcription factors also have specific roles in initiation-TAFsoTBP is associated with ten TAFs. oTwo TAFs bind DNA elements are the promoter (Inr and downstream elements)oSeveral TAFs have structural homology with histone proteins.-It has been proposed that they bind DNA similarly to histones.oAnother TAF regulates binding of TBP to DNA using an inhibitory flap displacement.-TFIIBoSingle polypeptide chainoEnters preinitiation complexoTFIIB-TBP-DNA complex shows contacts between TFIIB-TBP and TFIIB-DNA-These include base specific interactions with major and minor grooves.oThe asymmetric binding of TFIIB to TBBP-TATA accounts for the asymmetry in the rest of the assembly of the preinitiation complex and the unidirectional transcription that results. oAlso contacts Pol II in the preinitiation complex.oBridge TATA-bound TBP and polymerase.oStudies suggest that TFIIB inserts into RNA exit channel and active center cleft of Pol II.-Regions linker and reader aid in open complex formation by stabilizing the melted DNA until RNA:DNA hybrid takes over the role.-TFIIFoTwo subunit factor associates with Pol II and is recruited to the promoter with the enzyme.oBinding with Pol II stabilizes DNA TBP TFIIB complex and is required before TFIIE and TFIIH.-TFIIE and TFIIHoTFIIE has two subunits, binds next, and has roles in recruitment and regulation of TFIIH>oTFIIH controls ATP depending transition of preinitiation complex to the open complex.In Vivo, Transcription Initiation requires additional proteins, including the mediator complex-Requires transcriptional regulatory proteins, the mediator complex, and nucleosome modifying enzymes.-Additional requirements b/c DNA template is packed into chromatin, complicating binding to thepromoter of polymerase and associated factors. Mediator Consists of Many Subunits, Some Conserved from Yeast to Human-Yeast and human mediators each include more than 20 subunits.-Very few have identified functions. -Sr40/Med17 is essential for transcription of essentially all Pol II genes in vivo.-The Mediator from both is organized in modules, each with subset of subunits. oModules called head, middle (arm), and tail can be dissociated form one another under certain conditions in vitro.oVarious forms of mediator possible.-Different methods of isolation.-Different subsets of genes or responding to different groups of regulators (activators/repressors).A new set of factors stimulates Pol II elongation and RNA proofreading-Transition to elongation phaseoPol II enzyme shedding initiation factors (GTF and Mediator)oIn place, another set of factors (elongation factors) recruited.oCTD tail is very long-Allows to bind several components of elongation and processing machinery and deliver them to the emerging RNA.oProteins that stimulate elongation-P-TEFb-SPT5-ELL Family-TFIISElongating RNA Polymerase Must Deal with Histones in its Path-Chromatin greatly impedes transcription.-FACT - facilitates chromatin transcriptionElongating Polymerase is Associated with a New Set of Protein Factors Required for Various Types of RNAProcessing-Events: cap 5' end, splicing, polyadenylation of 3' end.-Splicing - where non-coding introns are removed to generate mature mRNA.-Capping oAddition of modified (methylated) guanine to 5' end.oJoined to the RNA transcript by unusual 5' - 5' linkage with three phosphates.oCreation of 5' cap-Phosphate group is removed from 5' end of transcript-GMP moiety is added-Nucleotide is modified by adding methyl group.oRNA is capped as soon as it emerges from exit channel.oDe-phosphorylation of Ser5 within tail repeats may be responsible for dissociation of thecapping machinery, and further phosphorylation causes recruitment of machinery for splicing.-PolyadenylationoLinked with termination of transcriptionoPolymerase CTD tail recruits enzymes necessaryoTwo protein complexes are carried by the CTD of polymerase as it approaches the end ofthe gene: CPSF (cleavage and polyadenylation specificity factor) and CSTF (Cleavage stimulation factor). The sequences that once transcribed into RNA trigger transfer of these factors to the RNA (poly A signal).