Chapter 31 Transcription and Regulation of Gene Expression to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham All 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
Outline • 31.1 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 mRNA
The 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 DNA • Polymerase 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 Eukaryotes • RNA 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 Prokaryotes • Genes for enzymes for pathways are grouped in clusters on the chromosome - called operons • This allows coordinated expression • A 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 genes
Induction and Repression • Increased 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 IPTG
The lac Operon • lacI mutants express the genes needed for lactose metabolism • The structural genes of the lac operon are controlled by negative regulation • lacI gene product is the lac repressor • The lac operator is a palindromic DNA • lac repressor - DNA binding on N-term; C-term. binds inducer, forms tetramer.
Catabolite Activator ProteinPositive Control of the lac Operon • Some promoters require an accessory protein to speed transcription • Catabolite Activator Protein or CAP is one such protein • CAP is a dimer of 22.5 kD peptides • N-term binds cAMP; C-term binds DNA • Binding of CAP-(cAMP)2 to DNA assists formation of closed promoter complex
The trp Operon • Encodes a leader sequence and 5 proteins that synthesize tryptophan • Trp repressor controls the operon • Trp repressor binding excludes RNA polymerase from the promoter • Trp repressor also regulates trpR and aroH operons and is itself encoded by the trpR operon. This is autogenous regulation (autoregulation).
Transcription Regulationin Eukaryotes • More complicated than prokaryotes • Chromatin limits access of regulatory proteins to promoters • Factors must reorganize the chromatin • In addition to promoters, eukaryotic genes have ‘enhancers’, also known as upstream activation sequences • DNA looping permits multiple proteins to bind to multiple DNA sequences