Bài giảng Biochemistry 2/e - Chapter 31: Transcription and Regulation of Gene Expression

Chapter 31Transcription and Regulation of Gene Expressionto accompanyBiochemistry, 2/ebyReginald Garrett and Charles GrishamAll rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777 Outline31.1 Transcription in Prokaryotes 31.2 Transcription in Eukaryotes 31.3 Regulation of Transcription in Prokaryotes 31.4 Transcription Regulation in Eukaryotes 31.5 Structural Motifs in DNA-Binding Proteins 31.6 Post-Transcriptional Processing of mRNAThe Postulate of Jacob and Monod Before it had been characterized in a molecular sense, messenger RNA was postulated to exist by F. Jacob and J. Monod.Their four properties: base composition that reflects DNA heterogeneous with respect to mass able to associate with ribosomes high rate of turnover Other Forms of RNA rRNA and tRNA only appreciated later All three forms participate in protein synthesis All made by DNA-dependent RNA polymerases This process is called transcription Not all genes encode proteins! Some encode rRNAs or tRNAs Transcription is tightly regulated. Only 0.01% of genes in a typical eukaryotic cell are undergoing transcription at any given moment How many proteins is that??? Transcription in Prokaryotes Only a single RNA polymerase In E.coli, RNA polymerase is 465 kD complex, with 2 , 1 , 1 ', 1  ' binds DNA  binds NTPs and interacts with   recognizes promoter sequences on DNA  subunits appear to be essential for assembly and for activation of enzyme by regulatory proteins Stages of Transcription See Figure 31.2 binding of RNA polymerase holoenzyme at promoter sites initiation of polymerization chain elongation chain termination Binding of polymerase to Template DNAPolymerase binds nonspecifically to DNA with low affinity and migrates, looking for promoter Sigma subunit recognizes promoter sequence RNA polymerase holoenzyme and promoter form "closed promoter complex" (DNA not unwound) - Kd = 10-6 to 10-9 M Polymerase unwinds about 12 pairs to form "open promoter complex" - Kd = 10-14 M Properties of Promoters See Figure 31.3 Promoters typically consist of 40 bp region on the 5'-side of the transcription start site Two consensus sequence elements: The "-35 region", with consensus TTGACA - sigma subunit appears to bind here The Pribnow box near -10, with consensus TATAAT - this region is ideal for unwinding - why? Initiation of Polymerization RNA polymerase has two binding sites for NTPs Initiation site prefers to binds ATP and GTP (most RNAs begin with a purine at 5'-end) Elongation site binds the second incoming NTP 3'-OH of first attacks alpha-P of second to form a new phosphoester bond (eliminating PPi) When 6-10 unit oligonucleotide has been made, sigma subunit dissociates, completing "initiation" Note rifamycin and rifampicin and their different modes of action (Fig. 31.4 and related text) Chain Elongation Core polymerase - no sigma Polymerase is accurate - only about 1 error in 10,000 bases Even this error rate is OK, since many transcripts are made from each gene Elongation rate is 20-50 bases per second - slower in G/C-rich regions (why??) and faster elsewhere Topoisomerases precede and follow polymerase to relieve supercoiling Chain Termination Two mechanisms Rho - the termination factor protein rho is an ATP-dependent helicase it moves along RNA transcript, finds the "bubble", unwinds it and releases RNA chain Specific sequences - termination sites in DNA inverted repeat, rich in G:C, which forms a stem-loop in RNA transcript 6-8 As in DNA coding for Us in transcript Transcription in EukaryotesRNA polymerases I, II and III transcribe rRNA, mRNA and tRNA genes, respectively Pol III transcribes a few other RNAs as well All 3 are big, multimeric proteins (500-700 kD) All have 2 large subunits with sequences similar to  and ' in E.coli RNA polymerase, so catalytic site may be conserved Pol II is most sensitive to -amanitin, an octapeptide from Amanita phalloides ("destroying angel mushroom") Transcription Factors More on this later, but a short note now The three polymerases (I, II and III) interact with their promoters via so-called transcription factors Transcription factors recognize and initiate transcription at specific promoter sequences Some transcription factors (TFIIIA and TFIIIC for RNA polymerase III) bind to specific recognition sequences within the coding region RNA Polymerase II Most interesting because it regulates synthesis of mRNA Yeast Pol II consists of 10 different peptides (RPB1 - RPB10) RPB1 and RPB2 are homologous to E. coli RNA polymerase  and ' RPB1 has DNA-binding site; RPB2 binds NTP RPB1 has C-terminal domain (CTD) or PTSPSYS 5 of these 7 have -OH, so this is a hydrophilic and phosphorylatable site More RNA Polymerase II CTD is essential and this domain may project away from the globular portion of the enzyme (up to 50 nm!) Only RNA Pol II whose CTD is NOT phosphorylated can initiate transcription TATA box (TATAAA) is a consensus promoter 7 general transcription factors are required See TFIID bound to TATA (Fig. 31.11) Transcription Regulation in ProkaryotesGenes for enzymes for pathways are grouped in clusters on the chromosome - called operonsThis allows coordinated expressionA regulatory sequence adjacent to such a unit determines whether it is transcribed - this is the ‘operator’Regulatory proteins work with operators to control transcription of the genesInduction and RepressionIncreased synthesis of genes in response to a metabolite is ‘induction’Decreased synthesis in response to a metabolite is ‘repression’Some substrates induce enzyme synthesis even though the enzymes can’t metabolize the substrate - these are ‘gratuitous inducers’ - such as IPTGThe lac OperonlacI mutants express the genes needed for lactose metabolismThe structural genes of the lac operon are controlled by negative regulationlacI gene product is the lac repressorThe lac operator is a palindromic DNAlac repressor - DNA binding on N-term; C-term. binds inducer, forms tetramer.Catabolite Activator ProteinPositive Control of the lac OperonSome promoters require an accessory protein to speed transcriptionCatabolite Activator Protein or CAP is one such proteinCAP is a dimer of 22.5 kD peptidesN-term binds cAMP; C-term binds DNABinding of CAP-(cAMP)2 to DNA assists formation of closed promoter complexThe trp OperonEncodes a leader sequence and 5 proteins that synthesize tryptophanTrp repressor controls the operonTrp repressor binding excludes RNA polymerase from the promoterTrp repressor also regulates trpR and aroH operons and is itself encoded by the trpR operon. This is autogenous regulation (autoregulation).Transcription Regulationin EukaryotesMore complicated than prokaryotesChromatin limits access of regulatory proteins to promotersFactors must reorganize the chromatin In addition to promoters, eukaryotic genes have ‘enhancers’, also known as upstream activation sequencesDNA looping permits multiple proteins to bind to multiple DNA sequencesStructural Motifs in DNA-Binding Regulatory Proteins Crucial feature must be atomic contacts between protein residues and bases and sugar-phosphate backbone of DNA Most contacts are in the major groove of DNA 80% of regulatory proteins can be assigned to one of three classes: helix-turn-helix (HTH), zinc finger (Zn-finger) and leucine zipper (bZIP) In addition to DNA-binding domains, these proteins usually possess other domains that interact with other proteins Alpha Helices and DNA A perfect fit! A recurring feature of DNA-binding proteins is the presence of -helical segments that fit directly into the major groove of B-form DNA Diameter of helix is 1.2 nm Major groove of DNA is about 1.2 nm wide and 0.6 to 0.8 nM deep Proteins can recognize specific sites in DNA The Helix-Turn-Helix Motif First identified in 3 prokaryotic proteins two repressor proteins (Cro and cI) and the E. coli catabolite activator protein (CAP) All these bind as dimers to dyad-symmetric sites on DNA (see Figure 31.33) All contain two alpha helices separated by a loop with a beta turn The C-terminal helix fits in major groove of DNA; N-terminal helix stabilizes by hydrophobic interactions with C-terminal helix Helix-Turn-Helix II See Figures 31.34 and 31.35 Residues 1-7 of the motif are the first helix (but called "helix 2") Residue 9 is the turn maker - a Gly, of course Residues 12-20 are the second helix (called "helix 3") Recognition of DNA sequence involves the sides of base pairs that face the major groove (see discussion on pages 1050-1052) The Zn-Finger Motif First discovered in TFIIIA from Xenopus laevis, the African clawed toad Now known to exist in nearly all organisms Two main classes: C2H2 and Cx C2H2 domains consist of Cys-x2-Cys and His-x3-His domains separated by at least 7-8 aas Cx domains consist of 4, 5 or 6 Cys residues separated by various numbers of other residues See Figure 31.37 and Table 31.7More Zn-Fingers Their secondary and tertiary structures C2H2 -type Zn fingers form a folded beta strand and an alpha helix that fits into the DNA major groove Cx-type Zn fingers consist of two mini-domains of four Cys ligands to Zn followed by an alpha helix: the first helix is DNArecognition helix, second helix packs against the first The Leucine Zipper Motif First found in C/EBP, a DNA-binding protein in rat liver nuclei Now found in nearly all organisms Characteristic features: a 28-residue sequence with Leu every 7th position and a "basic region" (What do you know by now about 7-residue repeats?) This suggests amphipathic alpha helix and a coiled-coil dimer The Structure of the Zipper and its DNA complex Leucine zipper proteins (aka bZIP proteins) dimerize, either as homo- or hetero-dimers The basic region is the DNA-recognition site Basic region is often modelled as a pair of helices that can wrap around the major groove Homodimers recognize dyad-symmetric DNA Heterodimers recognize non-symmetric DNA Fos and Jun are classic bZIPs Post-transcriptional Processing of mRNA in Eukaryotes Translation closely follows transcription in prokaryotes In eukaryotes, these processes are separated - transcription in nucleus, translation in cytoplasm On the way from nucleus to cytoplasm, the mRNA is converted from "primary transcript" to "mature mRNA" Eukaryotic Genes are Split Introns intervene between exons Examples: actin gene has 309-bp intron separates first three amino acids and the other 350 or so But chicken pro-alpha-2 collagen gene is 40-kbp long, with 51 exons of only 5 kbp total. The exons range in size from 45 to 249 bases Mechanism by which introns are excised and exons are spliced together is complex and must be precise Capping and Methylation Primary transcripts (aka pre-mRNAs or heterogeneous nuclear RNA) are usually first "capped" by a guanylyl group The reaction is catalyzed by guanylyl transferase Capping G residue is methylated at 7-position Additional methylations occur at 2'-O positions of next two residues and at 6-amino of the first adenine 3'-Polyadenylylation Termination of transcription occurs only after RNA polymerase has transcribed past a consensus AAUAAA sequence - the poly(A)+ addition site 10-30 nucleotides past this site, a string of 100 to 200 adenine residues are added to the mRNA transcript - the poly(A)+ tailpoly(A) polymerase adds these A residuesFunction not known for sure, but poly(A) tail may govern stability of the mRNA Splicing of Pre-mRNA Capped, polyadenylated RNA, in the form of a RNP complex, is the substrate for splicing In "splicing", the introns are excised and the exons are sewn together to form mature mRNA Splicing occurs only in the nucleus The 5'-end of an intron in higher eukaryotes is always GU and the 3'-end is always AG All introns have a "branch site" 18 to 40 nucleotides upstream from 3'-splice site Branch site is essential to splicing The Branch site and Lariat Branch site is usually YNYRAY, where Y = pyrimidine, R = purine and N is anything The "lariat" a covalently closed loop of RNA is formed by attachment of the 5'-P of the intron's invariant 5'-G to the 2'-OH at the branch A site The exons then join, excising the lariat.The lariat is unstable; the 2'-5' phosphodiester is quickly cleaved and intron is degraded in the nucleus.The Importance of snRNP Small nuclear ribonucleoprotein particles - snRNPs, pronounced "snurps" - are involved in splicing A snRNP consists of a small RNA (100-200 bases long) and about 10 different proteins Some of the 10 proteins are general, some are specific. Properties described on page 1063 snRNPs and pre-mRNA form the spliceosome Spliceosome is the size of ribosomes, and its assembly requires ATP Assembly of the Spliceosome See Figure 31.53 snRNPs U1 and U5 bind at the 5'- and 3'- splice sites, and U2 snRNP binds at the branch site Interaction between the snRNPs brings 5'- and 3'- splice sites together so lariat can form and exon ligation can occur The transesterification reactions that join the exons may in fact be catalyzed by "ribozymes"