Chapter 31. Transcription and RNA processing

Transcription

1 Chapter 31 Transcription and RNA processing

2 RNA polymerase (RNAP)

3 E. coli promoters

4 Components of E. coli RNA Polymerase Holoenzyme (α 2 ββ'ωσ)

5 Structure of prokaryotic RNAP

6 The closed and open state of RNAP The closed state The open state

7 Chain elongation

8 Transcriptional termination in prokaryotes Rho-independent termination Terminator sequence: Hairpin structure with oligo(u) tail Rho-dependent termination No obvious sequence similarity Rho factor: Helicase and NTPase activity

9 Terminator Poly(U) tail

10 Eukaryotic RNA polymerases RNA polymerase I (RNAP I, RNAP A) - rrna RNA polymerase II (RNAP II, RNAP B) - mrna RNA polymerase III (RNAP III, RNAP C) - trna, 5S rrna, small nuclear and cytosolic RNAs

11 RNA polymerase subunits

12 RNAP II elongation complex Roger Konberg (2001)

13 Eukaryotic promoters Mammalian RNA polymerase I has a bipartite promoter Core promoter elements (-31 to +6) Upstream promoter elements (-187 to -107) RNA polymerase II promoters GC box (Constitutive housekeeping genes) TATA box (-25 to -30, cell type-specific genes) CCAAT box (-70 to -90) Enhancers (selective gene expression) RNA polymerase III promoters Internal and/or upstream promoters

14 Eukaryotic transcription factors At least six general transcription factors (GTFs) Equivalent of prokaryotic σ factor GTFs form preinitiation complex (PIC) along with RNAP II and promoter DNA

15 Preinitiation complex (PIC)

16 Control of transcription in prokaryotes Efficiency of promoters Different σ factors Repressor Catabolite repression gene activation Attenuation Stringent response

17 The expression of the lac operon (Natural inducer) (Synthetic inducer)

18 Catabolite repression Adequate amount of glucose prevent the full expression of gene specifying proteins involving in the fermentation of numerous other catabolite, including lactose, arabinose, and galactose camp levels are low in the presence of glucose but rise when glucose become scarse Catabolite gene activator protein (CAP)-cAMP complex enhances the transcription of catabolite repressed operons

19 E. coli arabad operon

20 Attenuation

21 Operons subject to attenuation

22 Riboswitches Messenger RNAs that control gene expression through their ability to bind small molecule metabolites directly Regulate biosynthesis of thiamine pyrophosphate, vitamine B12, riboflavin, S-adenosylmethionine (SAM), guanine, adenine, lysine and so on TPP-dependent mrna conformations

23 Stringent response In stringent or poor growth conditions, cellular resources are diverted from growth and division to amino acid synthesis When there are no charged trna's available to bind to the ribosome, RelA synthesizes pppgpp and several ribosomal proteins convert pppgpp to ppgpp (p)ppgpp inhibits a number of energyconsuming cellular processes, including replication and transcription

24 Posttranscriptional processing Eukaryotic mrna processing 5 -Cap: 7-methylguanosine (m 7 G) and 2 -O-methylation Poly(A) tails Splicing rrna processing Prokaryotic rrna: cleavage of primary transcripts by several different RNases (and a few methylation) Eukaryotic rrna: Ribose and base methylation, and subsequent cleavage by RNases Self-splicing rrna: group I and II introns trna processing 5 -Cleavage by RNAse P 3 -Cleavage and trimming by many RNAse enzymes Attachment of 3 -CCA to eukaryotic trna primary transcripts Modified bases

25 5 -Cap of eukaryotic mrna

26 Processing of pre-mrna or heterogeneous nuclear RNAs (hnrnas)

27 The splicing reaction

28 Spliceosome Splicing takes place in the spliceosome Each spliceosome is composed of five small nuclear RNA proteins, called snrnps, and other protein factors

29 Alternative splicing: Multiple proteins from a single gene Alternative splicing in the rat α-topomyosin gene

30 RNA interferance (RNAi) Silencing of gene expression, triggered by the presence of double-stranded RNA homologous to portions of the gene Dicer cleaves the long double-stranded RNAs into short 21- to 25-base-pair small interfering RNAs (sirnas) sirnas and several proteins form RNA-induced silencing complex (RISC) Unzipping of double stranded sirna activates RISC, which can in turn bind to the target mrna and cleave it

31 RNA interferance (RNAi)

32 Posttrancriptional processing of E. coli rrna

33 Self-splicing rrna 2 Self-splicing rrna (Group I intron) of Tetrahymena thermophila

34 Small nucleolar RNAs (snornas) snornas guide the methylation or pseudouridylation of eukaryotic rrnas by complimentary base pairing C/D box: 2 -O-methylation H/ACA box: peudouridylation snornas and other nucleolar proteins form small nucleolar ribonucleoprotein (snornp)

35 trna processing Cleavage of 5 -end by RNAse P, whose RNA component has the catalytic function Many different RNase enzymes are involved in 3 - cleavage (or trimming) of prokaryotic initial trna transcripts The CCA ends of eukaryotic trnas are posttranscriptionally appended

36 Processing of eukaryotic trna Removal of 5 -extension by RNAse P 3 -CCA addition Modified bases Removal of intervening sequence (intron)