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Transcription Chapter 8 The Problem Information must be transcribed from DNA in order function further. REMEMBER: DNARNAProtein Tanscription in Prokaryotes Polymerization catalyzed by RNA polymerase Can initiate synthesis Uses rNTPs Requires a template Unwinds and rewinds DNA 4 stages Recognition and binding Initiation Elongation Termination and release RNA Polymerase 5 subunits, 449 kd (~1/2 size of DNA pol III) Core enzyme 2 subunits---hold enzyme together --- links nucleotides together ’---binds templates ---recognition Holoenzyme= Core + sigma RNA Polymerase Features Starts at a promoter sequence, ends at termination signal Proceeds in 5’ to 3’ direction Forms a temporary DNA:RNA hybrid Has complete processivity RNA Polymerase X-ray studies reveal a “hand” Core enzyme closed Holoenzyme open Suggested mechanism NOTE: when sigma unattached, hand is closed RNA polymerase stays on DNA until termination. Recognition Template strand Coding strand Promoters Core promoter elements for E. coli Binding sites for RNA pol on template strand ~40 bp of specific sequences with a specific order and distance between them. -10 box (Pribnow box) -35 box Numbers refer to distance from transcription start site Template and Coding Strands Sense (+) strand DNA coding strand Non-template strand 5’–TCAGCTCGCTGCTAATGGCC–3’ 3’–AGTCGAGCGACGATTACCGG–5’ transcription DNA template strand antisense (-) strand 5’–UCAGCUCGCUGCUAAUGGCC–3’ RNA transcript Typical Prokaryote Promoter Consensus sequences Pribnow box located at –10 (6-7bp) -35 sequence ~(6bp) Consensus sequences: Strongest promoters match consensus Up mutation: mutation that makes promoter more like consensus Down Mutation: virtually any mutation that alters a match with the consensus In Addition to Core Promoter Elements UP (upstream promoter) elements Gene activator proteins Ex. E. coli rRNA genes Facilitate recognition of weak promoter E. coli can regulate gene expression in many ways Stages of Transcription Template recognition Initiation Elongation RNA pol binds to DNA DNA unwound RNA pol moves and synthesizes RNA Unwound region moves Termination RNA pol reaches end RNA pol and RNA released DNA duplex reforms Transcription Initiation Steps Formation of closed promoter (binary) complex Formation of open promoter complex Ternary complex (RNA, DNA, and enzyme), abortive initiation Promoter clearance (elongation ternary complex) First rnt becomes unpaired Polymerase loses sigma NusA binds Ribonucleotides added to 3’ end Holoenzyme Core + Closed (Promoter) Binary Complex Open binary complex Ternary complex Promoter clearance Back Sigma () Factor Essential for recognition of promoter Stimulates transcription Combines with holoenzyme “open hand” conformation Positions enzyme over promoter Does NOT stimulate elongation Falls off after 4-9 nt incorporated “Hand” closes Variation in Sigma Variation in promoter sequence affects strength of promoter Sigmas also show variability Much less conserved than other RNA pol subunits Several variants within a single cell. EX: E. coli has 7 sigmas B. subtilis has 10 sigmas Different respond to different promoters Sigma Variability in E. coli Sigma70 (-35)CTGGCAC (-10)TTGCA alternative sigma factor involved in transcribing nitrogenregulated genes (among others). Sigma32 (-10)TATAAT Primary sigma factor, or housekeeping sigma factor. Sigma54 (-35)TTGACA (-35)TNNCNCNCTTGAA (-10)CCCATNT heat shock factor involved in activation of genes after heat shock. POINT: gives E. coli flexibility in responding to different conditions Sigma and Phage SP01 Early promoter—recognized by bacterial sigma factor. Transcription includes product, gp28. gp28 recognizes a phage promoter for expression of mid-stage genes, including gp33/34, which recognizes promoters for late gene expression. Promoter Clearance and Elongation Occurs after 4- 10 nt are added First rnt becomes unpaired from antisense (template) strand.DNA strands re-anneal Polymerase loses sigma, sigma recycled Result “Closed hand” surrounds DNA NusA binds to core polymerase As each nt added to 3’, another is melted from 5’, allowing DNA to re-anneal. RNA pol/NusA complex stays on until termination. Rate=20-50nt/second. Termination Occurs at specific sites on template strand called Terminators Two types of termination Intrinsic terminators Rho () dependent treminators Sequences required for termination are in transcript Variation in efficiencies. Intrinsic Terminators DNA template contains inverted repeats (G-C rich) Can form hairpins 6 to 8 A sequence on the DNA template that codes for U Consequences of poly-U:poly-A stretch? Coding strand Intrinsic Termination RNA pol passes over inverted repeats Hairpins begin to form in the transcript Poly-U:poly-A stretch melts RNA pol and transcript fall off UUUUU Rho () Dependent Terminators rho factor is ATP dependent helicase catalyses unwinding of RNA: DNA hybrid Rho Dependent Termination rho factor is ATP dependent helicase catalyzes unwinding of RNA: DNA hybrid 50~90 nucleotides/ sec (17 bp) Rho: Mechanism Rho binds to transcript at loading site (up stream of terminator) Hairpin forms, pol stalls Rho helicase releases transcript and causes termination hexamer Abortive initiation, elongation mRNA Function—Transcribe message from DNA to protein synthesis machinery Codons Bacterial—polycistronic Eukaryotic– monocistronic Leader sequence—non-translated at 5’ end May contain a regulatory region (attenuator) Also untranslated regions at 3’ end. Spacers (untranslated intercistronic sequences) Prokaryotic mRNA—short lived Eukaryotic mRNA-can be long lived Stable RNA rRNA -Structural component of ribosomes tRNA-Adaptors, carry aa to ribosome Synthesis Promoter and terminator Post-transcriptional modification (RNA processing) Evidence Both have 5’ monophospates Both much smaller than primary transcript tRNA has unusual bases. EX pseudouridine tRNA and rRNA Processing Both are excised from large primary transcripts 1º transcript may contain several tRNA molecules, tRNA and rRNA rRNAs simply excised from larger transcript tRNAs modified extensively 5. Base modifications Examples of Covalent Modification of Nucleotides in tRNA CH3 H H CH3 N N 6 N H N N 6 N N N C C CH3 CH2 O N N N6-Methyladenylate N6-Isopentenyladenylate (m6A) (i6A) O H H H H NH HN 3 4 N O Dihydrouridylate (D) 2 1 5 C H2C NH O N O Uridylate 5-oxyacetic acid (cmo5U) Pseudouridylate (Ψ) (ribose at C-5) N N 3 N CH3 6 CH2 5 NH NH 5 NH2 7-Methylguanylate (m7G) O O N N O 3-Methylcytidylate (m3C) O NH2 H3C N N O C NH 7 Inosinate (I) HO O N NH N N O CH3 O 5-Methylcytidylate (m5C) Base O H H O H O CH3 H 1' 2' 2’-O-Methylated nucleotide (Nm) Modifications are shown in blue. Eukaryotic Transcription Regulation very complex Three different pols Distinguished by -amanitin sensitivity Pol I—rRNA, least sensitive Pol II– mRNA, most sensitive Pol III– tRNA and 5R RNA moderately sensitive Each polymerase recognizes a distinct promoter Eukaryotic RNA Polymerases RNA Pol. Location Products -Amanitin Promoter Sensitivity I Nucleolus Large rRNAs (28S, 18S, 5.8S) II Nucleus Pre-mRNA, some snRNAs Highly sensitive III Nucleus tRNA, small rRNA (5S), snRNA Intermediate sensitivity Insensitive bipartite promoter Upstream Internal promoter and terminator Eukaryotic Polymerase I Promoters RNA Polymerase I Transcribes rRNA Sequence not well conserved Two elements Core element- surrounds the transcription start site (-45 to + 20) Upstream control element- between -156 and -107 upstream Spacing affects strength of transcription Eukaryotic Polymerase II Promoters Much more complicated Two parts Core promoter Core promoter Upstream element TATA box at ~-30 bases Initiator—on the transcription start site Downstream element-further downstream Many natural promoters lack recognizable versions of one or more of these sequences TATA-less Promoters Some genes transcribed by RNA pol II lack the TATA box Two types: Housekeeping genes ( expressed constitutively). EX Nucleotide synthesis genes Developmentally regulated genes. EX Homeotic genes that control fruit fly development. Specialized (luxury) genes that encode cell-type specific proteins usually have a TATA-box mRNA Processing in Eukaryotes Primary transcript much larger than finished product Precursor and partially processed RNA called heterogeneous nuclear RNA (hnRNA) Processing occurs in nucleus Splicing Capping Polyadenylation Capping mRNA 5’ cap is a reversed guanosine residue so there is a 5’-5’ linkage between the cap and the first sugar in the mRNA. Guanosine cap is methylated. First and second nucleosides in mRNA may be methylated BACK Polyadenylation Polyadenylation occurs on the 3’ end of virtually all eukaryotic mRNAs. Occurs after capping Catalyzed by polyadenylate polymerase Polyadenylation associated with mRNA half-life Histones not polyadenylated Introns and Exons Introns--Untranslated intervening sequences in mRNA Exons– Translated sequences Process-RNA splicing Heterogeneous nuclear RNA (hnRNA)-Transcript before splicing is complete Splicing Overview Occurs in the nucleus hnRNAs complexed with specific proteins, form a ribonucleoprotein particle (RNP) Primary transcripts assembled into hnRNP Splicing occurs on spliceosomes consist of Small nuclear ribonucleoproteins (SnRNPs) components of spliceosomes Contain small nuclear RNA (snRNA) Many types of snRNA with different functions in the splicing process Spliceosome Splice Site Recognition Introns contain invariant 5’-GU and 3’-AG sequences at their borders (GU-AG Rule) Recognized by small nuclear ribonucleoprotein particles (snRNPs) that catalyze the cutting and splicing reactions. Internal intron sequences are highly variable even between closely related homologous genes. Alternative splicing allows different proteins from a single original transcript Simplified Splicing Mechanism Close-up of Internal A Alternative Splicing I Exon removed with intron Alternative Splicing II Multiple 3’ cleavage sites EX. AG found at 5’ end of exon 2 and inside exon 2 RNA pol III Precursors to tRNAs,5SrRNA, other small RNAs Promoter Type I Lies completely within the transcribed region 5SrRNA promoter split into 3 parts tRNA promoters split into two parts Polymerase II-like promoters EX. snRNA Lack internal promoter Resembles pol II promoter in both sequence and position DNAse Footprinting Protected region Use: promoter ID End Label template strand Add DNA binding protein Digest with DNAse I Remove protein Separate on gel siRNA and microRNA Maxam-Gilbert Sequencing Prep ssDNA End label Treat with different reagents specific for each nt RNA Splicing RNA splicing is the removal of intervening sequences (IVS) that interrupt the coding region of a gene Excision of the IVS (intron) is accompanied by the precise ligation of the coding regions (exons) Eukaryotic Transcription 3 classes RNA pol (I-III) Many mRNA long lived 5’ and 3’ ends of mRNA modified. EX. 5’ cap Poly-A tail Primary mRNA transcript large, introns removed mRNA-monocistronic Eukaryotic RNA Polymerases RNA Pol. I II III Location Products -Amanitin sensitivity Nucleolus Large rRNAs (28S, 18S, 5.8S) Insensitive Nucleus Pre-mRNA, some snRNAs, snoRNAs Highly sensitive Nucleus tRNA, small rRNA (5S), snRNA Intermediate sensitivity Methods for Studying Regulatory DNA DNAse footprinting: (aka DNAse protection assay) identifies the sequence of DNA bound by a transcription factor protein binding prevents DNA from being attacked by DNAse I otherwise DNAse I cuts random sequences so that bands of many sizes are found on a gel everywhere EXCEPT where the transcription factor protects the DNA DNA Footprinting What are the roles of TAFs? TAFIIs help TBP with transcirption from promoters with initiators an downstream elemens • Prokaryotes • rrn operons •The bacterium have several rrn operons that contain rRNA genes. tRNA tRNA tRNA •Transcription of the rrnD operon yields a 30S precursor, which must be cut up to release the three rRNA and three tRNAs. •rRNA processing Formation of a cap at the 5’ end of a eukaryotic mRNA precursor 1 2 4 3 DNA footprinting short segment of 32P end-labeled dsDNA 1. Unprotected control DNA 2. Protected by DNA binding protein Partial digestion with DNase 1 Gelelectrophoresis and autoradiography 32P end-labeled fragments 2 Facts to remember DNA-dependent RNA synthesis: 1. 2. 3. 4. 5. 6. 7. 8. starts at a promoter sequence, ends at termination signal the first 5’-triphosphate is NOT cleaved proceeds in 5’ to 3’ direction new residues are added to the 3’ OH the template is copied in the 3’ – 5’ direction forms a temporary DNA:RNA hybrid transcription rate ( 50 to 90 nts/sec) RNA polymerase has complete processivity Rho (protein) dependent termination Is Rho a termination factor? Rho affects chain elongation, but not initiation. Rho causes production of shorter transcripts. Rho release transcripts from the DNA template. 1. 2. DNA termination site: sites on DNA with specific structure DNA template contains inverted repeats 6 to 8 A sequence on the DNA template that codes for U