Initiation and termination of transcription, Post transcription modification of the RNA. Mitesh Shrestha

Transcription

1 Initiation and termination of transcription, Post transcription modification of the RNA Mitesh Shrestha

2 Transcription: overview In prokaryotes transcription and translation are coupled. Proteins are synthesized directly from the primary transcript as it is made. In eukaryotes transcription and translation are separated. Transcription occurs in the nucleus, and translation occurs in the cytoplasm on ribosomes.

3 Transcription: RNA Polymerase DNA-dependent DNA template, ribonucleoside 5 triphosphates, and Mg 2+ Synthesizes RNA in 5 to 3 direction E. coli RNA polymerase consists of 5 subunits Eukaryotes have five RNA polymerases RNA polymerase II is responsible for transcription of protein-coding genes and some snrna molecules RNA polymerase II has 12 subunits Requires accessory proteins (transcription factors) Does not require a primer

4 The Process of Gene Expression For non-viral proteins Information stored in the nucleotide sequences of genes is translated into the amino acid sequences of proteins through unstable intermediaries called messenger (m)rnas. Synthesis of viral proteins in infected bacteria involved an unstable RNA molecule synthesized from the viral DNA.

5 RNA synthesis 1-Single strand of DNA (template strand) 2-ribonuleoside triphosphate (NTP) 3-no pre-existing primers (de novo) template strand=transcribed Protein

6 Nucleophilic attacks --3 hydroxyl group of the RNA strand --nucleotidyl phosphorus on the nucleoside triphosphate nucleotide, nucleoside monophosphate RNA polymerase Transcriptional factors

7 The Transcription Bubble Prokaryotes: --RNA polymerase binds specific nucleotide sequences (promoter regions) plus transcriptional factors --Single RNA polymerase --DNA unwinding (AT regions)

8 Eukaryotes: --several RNA polymerases --no direct recognition binding --transcriptional factors The Transcription Bubble

9 General Features of RNA Synthesis Similar to DNA Synthesis except The precursors are ribonucleoside triphosphates. Only one strand of DNA is used as a template. RNA chains can be initiated de novo (no primer required). The RNA molecule will be complementary to the DNA template (antisense) strand and identical (except that uridine replaces thymidine) to the DNA non-template (sense) strand. RNA synthesis is catalyzed by RNA polymerases and proceeds in the 5 3 direction.

10 In eukaryotes, genes are present in the nucleus, whereas polypeptides are synthesized in the cytoplasm. Messenger RNA molecules function as non-stable intermediaries that carry genetic information from DNA to the ribosomes, where proteins are synthesized. RNA synthesis, catalyzed by RNA polymerases, is similar to DNA synthesis in many respects. RNA synthesis occurs within a localized region of strand separation (Transcription Bubble), and only one strand of DNA functions as a template for RNA synthesis.

11 RNA synthesis, catalyzed by RNA polymerases, is similar to DNA synthesis in many respects. Prokaryotic: OriC (245 bp) AT-rich region (replication bubble) Eukaryotic: ARS (Autonomously Replicating Sequences) AT-rich region 11 bp

12 Stages of Transcription Promoter Recognition Chain Initiation Chain Elongation Chain Termination

13 Transcription: promoter recognition Transcription factors bind to promoter sequences and recruit RNA polymerase. DNA is bound first in a closed complex. Then, RNA polymerase denatures a bp segment of the DNA (open complex). The site where the first base is incorporated into the transcription is numbered +1 and is called the transcription start site. Transcription factors that are required at every promoter site for RNA polymerase interaction are called basal transcription factors.

14 Promoter recognition: promoter sequences Promoter sequences vary considerably. RNA polymerase binds to different promoters with different strengths; binding strength relates to the level of gene expression There are some common consensus sequences for promoters: Example: E. coli 35 sequence (found 35 bases 5 to the start of transcription) Example: E. coli TATA box (found 10 bases 5 to the start of transcription)

15 Properties of Promoters 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.

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17 Promoter recognition: enhancers Eukaryotic genes may also have enhancers. Enhancers can be located at great distances from the gene they regulate, either 5 or 3 of the transcription start, in introns or even on the noncoding strand. One of the most common ways to identify promoters and enhancers is to use a reporter gene.

18 Promoter recognition: other players Many proteins can regulate gene expression by modulating the strength of interaction between the promoter and RNA polymerase. Some proteins can activate transcription (upregulate gene expression). Some proteins can inhibit transcription by blocking polymerase activity. Some proteins can act both as repressors and activators of transcription.

19 Transcription: chain initiation Chain initiation: RNA polymerase locally denatures the DNA. The first base of the new RNA strand is placed complementary to the +1 site. RNA polymerase does not require a primer. The first 8 or 9 bases of the transcript are linked. Transcription factors are released, and the polymerase leaves the promoter region.

20 Transcription in Prokaryotes Transcription ---The first step in gene expression ---Transfers the genetic information stored in DNA (genes) into messenger (m)rna molecules that ---Carry the information to the sites of protein synthesis in the cytoplasm.

21 Stages of Transcription --DNA dependent RNA polymerase --5 to 3 direction --Walk (literally) on the DNA --Upstream and downstream regions

22 E. Coli RNA Polymerase Tetrameric core: 2 Holoenzyme: 2 (480,000 Daltons; bp~650 Daltons) Functions of the subunits: : assembly of the tetrameric core : ribonucleoside triphosphate binding site : DNA template binding region (sigma factor): initiation of transcription (*) (*) in vivo In vitro: RNA polymerase works just fine on both DNA strands

23 Initiation of RNA Chains Binding of RNA polymerase holoenzyme to a promoter region in DNA ( promoter region) Localized unwinding of the two strands of DNA by RNA polymerase to provide a single-stranded template Formation of phosphodiester bonds between the first few ribonucleotides in the nascent RNA chain

24 A Typical E. coli Promoter..,-2,-1,+1,+2,..

25 Numbering of a Transcription Unit The transcript initiation site is +1 (A/T). Bases preceding the initiation site are given minus ( ) prefixes and are referred to as upstream sequences. Bases following the initiation site are given plus (+) prefixes and are referred to as downstream sequences. Consensus sequences: highly conserved Recognition sequences: Sigma factor (

26 Transcription: chain elongation Chain elongation: RNA polymerase moves along the transcribed or template DNA strand. The new RNA molecule (primary transcript) forms a short RNA-DNA hybrid molecule with the DNA template.

27 Elongation Sigma factor needs to be released ---Re- and Un-winding activities -- Walk (literally) on the DNA 5 to 3 --growing RNA chain RNA polymerase binds both DNA template and growing RNA chain

28 Elongation phase of transcription Requires the release of RNA polymerase from the initiation complex Highly processive Dissociation of factors needed specifically at initiation. Bacterial dissociates from the holoenzyme Eukaryotic TFIID and TFIIA appear to stay behind at the promoter after polymerase and other factors leave the initiation complex

29 P-TEFb Proteins implicated in elongation Positive transcription elongation factor b Cyclin-dependent kinase Phosphorylates CTD of large subunit, Pol II E. coli GreA and GreB, eukaryotic TFIIS may overcome pausing by the polymerase induce cleavage of the new transcript, followed by release of the 3 terminal RNA fragment. E. coli NusG, yeast Spt5, human DSIF Regulated elongation (negative and positive), direct contact with polymerase and nascent transcript ELL: increase elongation rate of RNA Pol II CSB: Cockayne syndrome B protein, incr. elongation rate

30 Model for RNA Polymerase II Phosphorylation Eukaryotic RNA polymerase II Pol IIa kinase + ATP Pol IIo CTD of large subunit of Pol II CTD has repeat of (YSPTSPT) phosphatase P PPP P CTD of large subunit of Pol II P Model: Phosphorylation of Pol IIa to make Pol IIo is needed to release the polymerase from the initiation complex and allow it to start elongation.

31 The shift from initiation to elongation can be a regulated event. Release from pausing can be the mechanism for induction of expression. In Drosophila, the RNA polymerase can pause after synthesizing ~ 25 nucleotides of RNA in many genes. under elevated temperature conditions, the heat shock factor stimulates elongation by release from pausing. Other possible examples: mammalian c-myc, HIV LTR This is in addition to regulation at initiation.

32 Phosphorylated form of RNA PolII is at sites of elongation after heat shock Immunofluorescence Detection of Pol II on Drosophila Polytene Chromosomes. Green: dephosphorylated Red: hyperphosphorylated Yellow: mixed

33 Transcription: chain termination Most known about bacterial chain termination Termination is signaled by a sequence that can form a hairpin loop. The polymerase and the new RNA molecule are released upon formation of the loop.

34 Termination Signals in E. coli Rho-dependent terminators require a protein factor ( ) Rho-independent terminators do not require

35 Termination of transcription in E. coli: Rho-independent site 5'... U A G G C G U UA A U G G C G C C G C U U G C A C A A A G C C C U A A G A A U A A G G U U G+C rich region in stem U C G G G Run of U's 3' to stem-loop A U U U U U U...3'

36 Rho-independent terminators do not require intrinsic termination)

37 RNA transcription stops --when the newly synthesized RNA molecule forms a G-C-rich hairpin loop followed by a run of As --Create a mechanical stress --Pulls the poly-u transcript out of the active site of the RNA polymerase --A-U has very weak interaction

38 Termination of transcription in E. coli: Rho-dependent site 5'...AUCGCUACCUCAUAUCCGCACCUCCUCAAACGCUACCUCGACCAGAAAGGCGUCUCUU Termination occurs at one of these 3 nucleotides. Little sequence specificity: rich in C, poor in G. Requires action of rho ( ) in vitro and in vivo. Many (most?) genes in E. coli have rho-dependent terminators.

39 Termination Signals in E. coli Rho-dependent terminators (non-intrinsic) require a protein factor ( ) and rut site Rut proteins bind specific RNA sequences (>>Cs and <<<Gs) Not hairpins or other secondary Structures Rho utilization (rut) John Wiley & Sons, Inc.

40 Rho factor, or Rho is a hexamer, subunit size is 46 kda Is an RNA-dependent ATPase Is an essential gene in E. coli Rho binds to protein-free RNA and moves along it (tracks) Upon reaching a paused RNA polymerase, it causes the polymerase to dissociate and unwinds the RNA-DNA duplex, using ATP hydrolysis. This terminates transcription.

41 Model for action of rho factor hexamer binds to protein-free RNA and moves along it. ' -dependent site RNA polymerase transcribes along the template, and moves along the RNA. RNA polymerase pauses at the -dependent terminator site, and catches up Structure in RNA that causes pausing unwinds the RNA-DNA hybrid and transcription terminates

42 mrna Structure in Bacteria : Coupling Transcription Termination and Translation lacz lacy laca Genes in operon transcription AUG UAA AUG UAA AUG UAA Polycistronic mrna translation -galactosidase lactose permease -galactoside transacetylase

43 Translation can occur simultaneously with transcription in bacteria lacz lacy laca AUG UAA Transcription of genes Nascent polypeptide ribosome Translation of mrna -galactosidase

44 Coupled Transcription and Translation in E. coli John Wiley & Sons, Inc.

45 Polarity Polar mutations occur in a gene early in an operon, but affect expression of both that gene and genes that follow in the operon. Usually affect translation at the beginning of an operon, and exert a negative effect on the transcription of genes later in the operon. Usually are nonsense (translation termination) mutations in a 5 gene that cause termination of transcription of subsequent genes in the operon. Rho mutants can suppress polarity.

46 lac Z lac Y lac A Diagram of polar wt: txn tln -galactosidase permease Ac'ase effects missense mutation: txn x x tln permease Ac'ase no -galactosidase activity nonsense mutation: txn Stop tln x x tln -dependent terminator of txn no -galactosidase protein (truncated protein gets degraded) no permease no Ac'ase

47 Model for involvement of rho in polar effects of nonsense mutations Wild-type -dependent site within a transcripton unit Ribosomes prev ent from catching up with RNA polymerase ribosome Structure in RNA that causes pausing Nonsense mutation nonsens e mutation nonsense Ribosomes dissociate at nonsense codon Structure in RNA that causes pausing Transcription and translation continues past the dependent termination site.

48 Eukaryotic mrna structure

49 Transcription and RNA Processing in Eukaryotes Five different enzymes catalyze transcription in eukaryotes, and the resulting RNA transcripts undergo three important modifications, including the excision of noncoding sequences called introns. The nucleotide sequenced of some RNA transcripts are modified post-transcriptionally by RNA editing.

50 Modifications to Eukaryotic pre-mrnas A 7-Methyl guanosine cap is added to the 5 end of the primary transcript by a 5-5 phosphate linkage. ( stability and protection) A poly(a) tail (a nucleotide polyadenosine tract, As) is added to the 3 end of the transcript. The 3 end is generated by cleavage rather than by termination. (stability and protection) When present, intron sequences are spliced out of the transcript. (stability)

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52 Eukaryotes Have Five RNA Polymerases RNA polymerase II Nucleus mirna Pre-mRNA~Heterogeneous nuclear RNA (hnrna)

53 A Typical RNA Polymerase II Promoter (mrna) Promoter: short sequence of conserved elements (seq. of DNA) located upstream from the transcript starting point. --~200 bp ( DNA linear) --~10 Kdp ( DNA bending)

54 Initiation by RNA Polymerase II Transcriptional factors (proteins) --Help/modulate/assist Basal transcriptional factors --bind close to the transcript starting point Other factors (enhancers and silencers) -TFIID --TATA-biding proteins (TBP) -TFIIA -TFIIB

55 -TFIIF (together with the RNA Pol-II) --enzymatic activity (DNA-unwinding) -TFIIE --binds downstream regions -TFIIH (helicase activity) -TFIIJ --binds downstream regions Helicase activity: it separates two annealed nucleic acid strands RNA Pol-II DNA-unwinding activity: RNA Polymerase bends and wraps around DNA. TFIIF alters (nonspecific) DNA binding by RNA polymerase II, resulting in substantial DNA unwinding but not DNA strand separation.

56 -TFIIH (helicase activity and kinase activity) When RNA polymerase II binds to the complex, it initiates transcription. Phosphorylation of the CTD is required for elongation to begin. CTD: carboxy-terminal domain

57 All eukaryotic RNA polymerases have 12 subunits and are aggregates of >500 kd. (nucleotide pair~0.660 kd) Some subunits are common to all three RNA polymerases. The largest subunit in RNA polymerase II has a CTD (carboxy-terminal domain) consisting of multiple repeats of a heptamer. -Typical RNA polymerase isolated from yeast (S. cerevisiase) ( and subunits) - subunits: CTD carboxy-terminal domain, which consists in multiple repeats of 7 amino acids, unique and important of regulation (tyrosine (Try, Y), serine (Ser, S) and threonine (Thr, T) residues) -Some subunits are common to all three polymerases. Figure 24.2

58 RNA Polymerase I Has a Bipartite Promoter The RNA polymerase I promoter consists of: --a core promoter --an upstream control element (UPE) RNA Pol I transcribes rrna genes. Core promoter: -45 to +20 seq., G-C-rich and A-T-rich (Inr-initiator) regions, Binding factors - protein complexes formed by TFIs and TBP-(TATA binding protein)

59 RNA Polymerase III Uses Both Downstream and Upstream Promoters RNA polymerase III has (3) types of promoters. -RNA Pol III transcribes trna -Core promoters (boxes) -Transcriptional Factors(TF) III: general and specifics * Figure 24.7 *proximal sequence element

60 RNA Chain elongation --Model John Wiley & Sons, Inc.

61 The 7-Methyl Guanosine (7-MG) Cap Histones:? FACT (facilitates chromatin transcriptional) Energy John Wiley & Sons, Inc.

62 RNA Chain termination Termination signal: specific DNA seq to 2000 nucleotides The 3 Poly(A) Tail --AAUAAA seq. --GU-rich seq. Endonuclease --poly(a) polymerase Pol-II vs Pol I and III -Terminator proteins (Rho-indep. Terminator)

63 Termination of transcription in eukaryotes : Pol I Termination by RNA polymerase I requires a binding site for a protein, Reb1p, that causes pausing. Model for Pol I termination. RNA polymerase I Reb1p U-rich If the Reb1p binding site in the DNA is replaced with the binding site for E. coli Lac repressor, Lac repressor protein will induce termination in an in vitro transcription reaction.

64 Termination of transcription in eukaryotes : Pol II and Pol III RNA polymerase III terminates in a run of 4-5 T s on the nontemplate strand, surrounded by G+C-rich DNA. No clear evidence for a discrete terminator of transcription by RNA polymerase II. The 3 end of the mrna is made by cleavage and polyadenylation.

65 Transcription: mrna synthesis/processing Prokaryotes: mrna transcribed directly from DNA template and used immediately in protein synthesis Eukaryotes: primary transcript must be processed to produce the mrna Noncoding sequences (introns) are removed Coding sequences (exons) spliced together 5 -methylguanosine cap added 3 -polyadenosine tail added

66 Transcription: mrna synthesis/processing Removal of introns and splicing of exons can occur several ways For introns within a nuclear transcript, a spliceosome is required. Splicesomes protein and small nuclear RNA (snrna) Specificity of splicing comes from the snrna, some of which contain sequences complementary to the splice junctions between introns and exons Alternative splicing can produce different forms of a protein from the same gene Mutations at the splice sites can cause disease Thalassemia Breast cancer (BRCA 1)

67 Transcription: mrna synthesis/processing RNA splicing inside the nucleus on particles called spliceosomes. Splicesomes are composed of proteins and small RNA molecules ( bp; snrna). Both proteins and RNA are required, but some suggesting that RNA can catalyze the splicing reaction. Self-splicing in Tetrahymena: the RNA catalyzes its own splicing Catalytic RNA: ribozymes

68 RNA Editing Usually the genetic information is not altered in the mrna intermediary. Sometimes RNA editing changes the information content of genes by Inserting or deleting uridine monophosphate residues. Changing the structures of individual bases

69 Editing of Apoplipoprotein-B mrna (Amino groups)

70 Three to five different RNA polymerases are present in eukaryotes, and each polymerase transcribes a distinct set of genes. Eukaryotic gene transcripts usually undergo three major modifications: the addition of 7-methyl guanosine caps to 5 termini, The addition of poly(a) tails to 3 ends, The information content of some eukaryotic transcripts is altered by RNA editing, which changes the nucleotide sequences of transcripts prior to their translation.

71 Interrupted Genes in Eukaryotes: Exons and Introns Most eukaryotic genes contain noncoding sequences called introns that interrupt the coding sequences, or exons. The introns are excised from the RNA transcripts prior to their transport to the cytoplasm.

72 Hybridization: annealing

73 R-Loop Evidence of an Intron in the Mouse -Globin Gene mrna Missing in actions Pre-mRNA

74 Introns Introns (or intervening sequences) are noncoding sequences located between coding sequences. Introns are removed from the pre-mrna and are not present in the mrna. Introns are variable in size and may be very large ( 50 bp to 3000 bp). Exons (both coding and noncoding sequences) are composed of the sequences that remain in the mature mrna after splicing. The biological significance of introns is still open to debate.

75 Removal of Intron Sequences by RNA Splicing The noncoding introns are excised from gene transcripts by several different mechanisms. Eukaryotes No prokaryotes (excepts a few a prokaryotes virus and others)

76 Conserved seq. for mrna Exon-GT AG-exon intron 99% Excision of Intron Sequences Ribonucleoproteins: Spliceosomes (1981)

77 Splicing Removal of introns must be very precise. Conserved sequences for removal of the introns of nuclear mrna genes are minimal. Dinucleotide sequences at the 5 and 3 ends of introns. Exon-GU AG-exon TACTAAC box (branch site with A) about 30 nucleotides upstream from the 3 splice site.

78 Spliceosomes: snrna plus ~40 proteins 1%: CG AG AT AC

79 Nuclear splicing involves trans-esterification GU UACUAAC.AG Branch site

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81 trna A splicing endonuclease makes two cuts at the end of the intron. A splicing ligase joins the two ends of the trna to produce the mature trna. Specificity resides in the three-dimensional (secundary) structure of the trna precursor, not in the nucleotide sequence.

82 rrna (Autocatalytic Splicing) G-3 -OH: absolute requirement Co-factor

83 Noncoding intron sequences are excised from RNA transcripts in the nucleus prior to the transport to the cytoplasm. Introns in trna precursors are removed by the concerted action of a splicing endonuclease and ligase, whereas introns in some rrna precursors are spliced out autocatalytically with no catalytic protein involved.

84 The introns in nuclear pre-mrnas are excised on complex ribonucleoprotein structures called spliceosomes. The intron excision process must be precise, with accuracy to the nucleotide level, to ensure that codons in exons distal to introns are read correctly during translation.

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