BCHM 4116 1st EditionExam# 3 Study Guide Lectures: 22 - 31Chapter 29: Transcription and Regulation of Gene Expression1. 29.1 – Genes Transcribed in Prokaryotes a. RNA poli.α2ββ ' σii.β β' = 2 large subunits1. performs most of enzymatic function iii. Binding of σ - subunit recognizes diff. DNA sequences that act as promoters1. Promoters = identify transcription start site b. Transcription has 4 stagesi. Binding of RNA pol at promter site1.σ – subunit recognizes promoter sequence2.RNA pol∧ promter formacomplex3. AKA “closed” promoter complex b/c dsDNA is not “opened” 4. +1 = transcription start site 5. within promoter are 2 consensus sequencesa. Pribnow box -10 region i. TATAATb. -35 region 6.σ – subunit recognizes and binds to -10 & -35 regions ii. Initiation of polymerization1. RNA pol does not require primer 2.σ – subunit dissociates from RNA pol once 9-12 resides long has been formed = completion of initiation 3.core RNA pol = highly processive and goes on to synthesize remainder of mRNAiii. Chain elongation1. Catalyzed by core polymerase 2. To prevent torsional stress from inhibiting transcription, gyrase introduces negative supercoils (& thereby remove positive supercoils) ahead of RNA pol3. Topoisomerase removes negative supercoil behind DNA iv. Chain termination 1. Bacteriaa. Intrinsic terminationi. Specific sequences in DNA called termination sites 1. Inverted repeats, typically G-C rich,so forms stem-loop structure 2. Nonrepeating segment that punctuates inverted repeats3. 6-8 A’s in DNA, coding for U’s in transcriptb. Rho factori. Less common & mechanistically more complexii. ATP-dependent hexameric helicaseiii. Catalyzes unwinding of RNA:DNA duplexiv. Rho factor recognizes and binds to C-rich region , then advances in 5’3’ direction until reaches transcription bubble v. There it catalyzes unwinding of transcript and template, releasing RNA chain 2. Regulation of Transcription in Prokaryotesa. Operator: regulatory sequence lying adjacent to DNA being transcribed determines whether transcription takes placeb. Operons: transcriptional control regions & a set of related structural genes, all organized in a contiguous linear array along chromosome i. Expression of operon is determined by access of RNA pol to promoter and occupancy of operation by regulatory proteins influences this accessii. Induction activates transcription from promoter; repression prevents it c. Attenuation regulates transcription after it has beguni. Any regulation mechanism that manipulates transcription termination or transcription pausing to regulate gene transcription downstream3. 29.3 – Gene Transcription in Eukaryotesa. DNA of eukaryotes is wrapped around histones forming nucleosomesb. Nucleosomes repress gene expressionc. Nucleosomes control gene expression by controlling transcriptional apparatusi. 2 classes of transcriptional co-regulators necessary to overcome nucleosome repression:1. Enzymes that covalently modify nucleosome histone proteins to loosen histone-DNA interactions2. ATP-dependent chromatin-remodeling complexesd. Transcription in Eukaryotes:e. The three classes of RNA polymerase in eukaryotes: RNA polymerases I, II, and III i. RNA pol I = localized to nucleolus & transcribes rRNAii. RNA pol II transcribes protein-encoding genes & mRNAiii. RNA pol III – transcribes tRNA genes, rRNA genes, other small RNAsiv. ALL 3 RNA pol interact w/ their promoters via transcription factors 1. Transcription factors: DNA-binding proteins that recognize & accurately initiate transcription at specific promoter sequencesf. The structure and function of RNA polymerase II, the mRNA-synthesizing RNA polymerase i. CTD is essential to RNA pol II functionii. Only RNA pol II whose CTD is not phosphorylated can initiate transcription1. But phosphorylation = transcription elongation 2. Phosphorylation triggers conversion of an initiation complex into an elongation complex g. Transcription regulation in eukaryotes, including: i. General features of gene regulatory sequences: promoters, enhancers, and response elements 1. Promoters consist of 2 features:a. Core element, near transcription start site where general transcription factors bindb. Regulatory elements 2. Enhancers assist initiationa. Location of enhancers relative to transcription start site is not fixedi. May be thousand nucleotides away from promoterii. Act to enhance transcription even if positioned downstreamb. BIDIRECTIONAL in that they function in either orientation c. “promiscuous” b/c they stimulate transcription from an promoter that happens to be in their vicinity d. enhancer function is dependent on recognition by a specific transcription factor 3. Response Elements : promoter responsive to common regulation a. Ex. heat shock element (HSE)b. Glucocorticoid response element (GRE_) c. Found in promoter region ii. Transcription initiation by RNA pol II requires TBP and GTFs1. The general transcription factors (GTFs) a. GTFs = TFIIA, TFIIB, TFIID, TFIIF, TFIIH, TFIISb. TFIID – TBPc. TFIIA – stabilizes TFIID and TATA boxd. TFIIB – recruits preinitiation complex (PIC) to promoter, positioning RNA pol active site over promotere. TFIIE & TFIIF – enhance promoter: PIC interaction f. TFIIH – helicase, aids in creating transcription bubble2. Mediatorsa. Bridge b/w enhancers & transcription co-activators to RNA pol at the promoterb. Ultimate regulator of transcription c. Acts as repressor and activator of transcription3. Alleviating the repression due to nucleosomes a. Relief from repression imposed by chromatin structure requires factors that can reorganize chromatin & alter nucleosomes so that promoters become accessible to transcriptional machinery i. Chromatin-remodeling complexes1. Mediate ATP-dependent conformational noncovalent changes in nucleosome structures2. Nucleic acid stimulated multisubunit ATPaseii. Histone-modifying enezymes1. Introduce covalent modifications into N-terminal tails of histone a. 1. Diminish DNA:histone associations via disruption of electrostatic interactionsb. 2. Introduce subsititutaions that can recruit binding of new protein participants through protein-protein interactionsb. Histone acetyl transferases (HATs)i. Acetylation of amino group on lysine residues in histone tails ii. N-acetylation suppresses positive charge in histone tails, diminishing their interaction w/ negatively charged DNAc. Transcription regulation: i. Phosphorylation of Ser residues ii.