Transcription

1. Transcription

2. Central Dogma

3. Genes • Sequence of DNA that is transcribed. • Encode proteins, tRNAs, rRNAs, etc.. • “Housekeeping” genes encode proteins or RNAs that are essential for normal cellular activity. • Simplest bacterial genomes contain 500 to 600 genes. • Mulitcellular Eukaryotes contain between 15,000 and 50,000 genes.

4. Types of RNAs • tRNA, rRNA, and mRNA • rRNA and tRNA very abundant relative to mRNA. • But mRNA is transcribed at higher rates than rRNA and tRNA • Abundance is a reflection of the relative stability of the different forms of RNA

5. RNA Content of E. coli Cells

6. Phases of Transcription • Initiation: Binding of RNA polymerase to promoter, unwinding of DNA, formation of primer. • Elongation: RNA polymerase catalyzes the processive elongation of RNA chain, while unwinding and rewinding DNA strand • Termination: termination of transcription and disassemble of transcription complex.

7. E. Coli RNA Polymerase • RNA polymerase core enzyme is a multimeric protein a2,b, b’, w • The b’ subunit is involved in DNA binding • The b subunit contains the polymerase active site • The a subunit acts as scaffold on which the other subunits assemble. • Also requires s-factor for initiation –forms holo enzyme complex Site of DNA binding and RNA polymerization

8. s-factor • The s-factor is required for binding of the RNA polymerase to the promoter • Association of the RNA polynerase core complex w/ the s-factor forms the holo-RNA polymerase complex • W/o the s-factor the core complex binds to DNA non-specifically. • W/ the s-factor, the holo-enzyme binds specifically with high affinity to the promoter region • Also decreases the affinity of the RNA polymerase to non-promoter regions • Different s-factors for specific classes of genes

9. Promoter Transcribed region terminator 5’ 3’ General Gene Structure • Promoter – sequences recognized by RNA polymerase as start site for transcription. • Transcribed region – template from which mRNA is synthesized • Terminator – sequences signaling the release of the RNA polymerase from the gene.

10. Gene Promoters • Site where RNA polymerase binds and initiates transcription. • Gene that are regulated similarly contain common DNA sequences (concensus sequences) within their promoters

11. Important Concensus Sequences • Pribnow Box – position –10 from transcriptional start • -35 region – position –35 from transcriptional start. • Site where s70-factor binds.

12. Other s-Factors • Standard genes – s70 • Nitrogen regulated genes – s54 • Heat shock regulated genes – s32

13. How does RNA polymerase finds the promoter? • RNA polymerase does not disassociate from DNA strand and reassemble at the promoter (2nd order reaction – to slow) • RNA polymerase holo-enzyme binds to DNA and scans for promoter sequences (scanning occurs in only one dimension, 100 times faster than diffusion limit) • During scanning enzyme is bound non-specifically to DNA. • Can quickly scan 2000 base pairs

14. Transcriptional Initiation • Rate limiting step of trxn. • Requires unwinding of DNA and synthesis of primer. • Conformational change occurs after DNA binding of RNA polymerase holo-enzyme. • First RNA Polymerase binds to DNA (closed-complex), then conformational change in the polymerase (open complex) causes formation of transcription bubble (strand separation).

15. 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“ • NusA protein binds to core complex after disassociation of s-factor to convert RNA polymerase to elongation form.

16. Transcriptional Initiation Closed complex Open complex Primer formation Disassociation of s-factor

17. 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

19. Transcriptional Termination • Process by which RNA polymerase complex disassembles from 3’ end of gene. • Two Mechanisms – Pausing and “rho-mediated” termination

20. Pausing induces termination • RNA polymerase can stall at “pause sites” • Pause sites are GC rich (difficult to unwind) • Can decrease trxn rates by a factor of 10 to 100. • Hairpin formation in RNA can exaggerate pausing • Hairpin structures in transcribed RNA can destabilize DNA:RNA hybrid in active site • Nus A protein increases pausing when hairpins form. 3’end tends to be AU rich easily to disrupt during pausing. Leads to disassembly of RNA polymerase complex

21. Rho Dependent Termination • rho is an ATP-dependent helicase • it moves along RNA transcript, finds the "bubble", unwinds it and releases RNA chain

23. Eukaryotic Transcription • Similar to what occurs in prokaryotes, but requires more accessory proteins in RNA polymerase complex. • Multiple RNA polymerases

24. Eukaryotic RNA Polymerases

25. Eukaryotic RNA Polymerases • RNA polymerase I, II, and III • 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

26. Eukaryotic Gene Promoters • Contain AT rich concensus sequence located –19 to –27 bp from transcription start (TATA box) • Site where RNA polymerase II binds

27. 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

28. 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

29. Transcription Factors • Polymerase I, II, and III do not bind specifically to promoters • They must interact with their promoters via so-called transcription factors • Transcription factors recognize and initiate transcription at specific promoter sequences

30. Transcription Factors • TFAIIA, TFAIIB – components of RNA polymerase II holo-enzyme complex • TFIID – Initiation factor, contains TATA binding protein (TBP) subunit. TATA box recognition. • TFIIF – (RAP30/74) decrease affinity to non-promoter DNA

31. Eukaryotic Transcription • Once initiation complex assembles process similar to bacteria (closed complex to open complex transition, primer formation) • Once elongation phase begins most transcription factor disassociate from DNA and RNA polymerase II (but TFIIF may remain bound). • TFIIS – Elongation factor binds at elongation phase. May also play analogous role to NusA protein in termination.

32. Transcriptional Regulation and RNA Processing

33. Gene Expression • Constitutive – Genes expressed in all cells (Housekeeping genes) • Induced – Genes whose expression is regulated by environmental, developmental, or metabolic signals.

34. Regulation of Gene Expression RNA Processing mRNA RNA Degradation 5’CAP AAAAAA Active enzyme Post-translational modification Protein Degradation

35. Transcriptional Regulation • Regulation occurring at the initiation of transcription. • Involves regulatory sequences present within the promoter region of a gene (cis-elements) • Involves soluble protein factors (trans-acting factors) that promote (activators) or inhibit (repressors) binding of the RNA polymerase to the promoter

36. Cis-elements • Typically found in 5’ untranscribed region of the gene (promoter region). • Can be specific sites for binding of activators or repressors. • Position and orientation of cis element relative to transcriptional start site is usually fixed.

37. Enhancers • Enhancers are a class of cis-elements that can be located either upstream or downstream of the promoter region (often a long distance away). • Enhancers can also be present within the transcribed region of the gene. • Enhancers can be inverted and still function 5’-ATGCATGC-3’ = 5’-CGTACGTA-3’

39. Two Classes of Trans-Acting Factors • Activators and repressors- Bind to cis-elements. • Co-activators and co-repressors – bind to proteins associated with cis-elements. Promote or inhibit assembly of transcriptional initiation complex

40. Structural 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

41. The Helix-Turn-Helix Motif • 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

42. The Zn-Finger Motif Zn fingers form a folded beta strand and an alpha helix that fits into the DNA major groove.

43. The Leucine Zipper Motif • Forms amphipathic alpha helix and a coiled-coil dimer • Leucine zipper proteins dimerize, either as homo- or hetero-dimers • The basic region is the DNA-recognition site • Basic region is often modeled as a pair of helices that can wrap around the major groove

44. Binding of some trans-factors is regulated by allosteric modification

45. 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

46. Induction and Repression • Increased synthesis of genes in response to a metabolite is ‘induction’ • Decreased synthesis in response to a metabolite is ‘repression’

47. lac operon • Lac operon – encodes 3 proteins involved in galactosides uptake and catabolism. • Permease – imports galactosides (lactose) • b-galactosidase – Cleaves lactose to glucose and galactose. • b-galactoside transacetylase – acetylates b-galactosides • Expression of lac operon is negatively regulated by the lacI protein

48. The lac I protein • The structural genes of the lac operon are controlled by negative regulation • lacI gene product is the lac repressor • When the lacI protein binds to the lac operator it prevents transcription • lac repressor – 2 domains - DNA binding on N-term; C-term. binds inducer, forms tetramer.

49. Inhibition of repression of lac operon by inducer binding to lacI • Binding of inducer to lacI cause allosteric change that prevents binding to the operator • Inducer is allolactose which is formed when excess lactose is present.

50. Catabolite Repression of lac Operon (Positive regulation) • When excess glucose is present, the lac operon is repressed even in the presence of lactose. • In the absence of glucose, the lac operon is induced. • Absence of glucose results in the increase synthesis of cAMP • cAMP binds to cAMP regulatory protein (CRP) (AKA CAP). • When activated by cAMP, CRP binds to lac promoter and stimulates transcription.