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Chapter 31

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Chapter 31

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  1. 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

  2. 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

  3. 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

  4. 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???

  5. 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

  6. Stages of Transcription See Figure 31.2 • binding of RNA polymerase holoenzyme at promoter sites • initiation of polymerization • chain elongation • chain termination

  7. 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

  8. 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?

  9. 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)

  10. 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

  11. 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

  12. 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")

  13. 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

  14. 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

  15. 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)

  16. 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

  17. 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

  18. 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.

  19. 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

  20. 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).

  21. 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