Transcription PDF

Transcription  CHAPTER OBJECTIVES 1. Describe the features of transcription 2. Discuss the requirements and steps in transcription 3. Explain the different mechanisms in RNA processing Transmission of Genetic Information Universal Features of Transcription 1. Transcription is a...

Transcription  CHAPTER OBJECTIVES 1. Describe the features of transcription 2. Discuss the requirements and steps in transcription 3. Explain the different mechanisms in RNA processing Transmission of Genetic Information Universal Features of Transcription 1. Transcription is a DNA-dependent synthesis of RNA. Template: DNA ( ) Product: 1. Messenger RNA (mRNA) 2. Ribosomal RNA (rRNA) 3. Transfer RNA (tRNA) 4. miRNA, siRNA and other regulatory RNAs 2. It involves certain regions of DNA. (vs. replication) DNA sequences that code for RNA do not code for an RNA  Universal Features of Transcription 3. It consists of 3 general steps. initiation, elongation, termination 4. It is the major point of control of gene expression. 5. The main enzyme for transcription is. Goodsell, D. The Machinery of Life. 2nd Ed.  Definition of Some Terms ntroianstpcir unti - basic unit of a gene which extends from promoter to terminator  Definition of Some Terms pmyriar tarnsirctp - immediate product of RNA polymerase before any changes occur to the RNA - regions close to promoter - regions farther away from promoter distal proximal promoter proximal distal  Definition of Some Terms (+1) - base pair of DNA which corresponds to the first RNA nucleotide added by RNA polymerase - DNA sequence from startpoint to any nucleotide(s) located towards the direction of transcription nucleotide position # is (+) - DNA sequence from startpoint to any nucleotide(s) located opposite/away from the direction of transcription nucleotide position # is (-)  Definition of Some Terms promoter -3 +1 +7 Upstream Downstream  Definition of Some Terms - nucleotide sequence which when aligned with each other have certain nucleotides that at certain positions occur at high frequency consensus seq: gcgcgcaggcgc  RNA Polymerase ▪ DNA-directed ▪ adds ribonucleotide units to the 3’OH end of the RNA chain → builds RNA in the direction ▪ can initiate the synthesis of strands on DNA templates ▪ error made for every 104-106 ribonucleotides incorporated ▪ no proofreading ability ▪ Prokaryotic vs Eukaryotic  Bacterial RNA Polymerase Subunits Subunit Gene Possible Role 2α rpoA aids in promoter recognition β rpoC binds ribonucleotide substrate β' rpoB binds DNA template σ rpoD recognizes and binds tightly to promoter ω facilitate assembly 2αββ’= core enzyme (2αββ’)σ = holoenzyme Bacterial RNA Polymerase rifampicin o binds to of bacterial RNA pol o prevents initiation of transcription  Eukaryotic RNA Polymerases Type Location #/cell Genes transcribed % activity I nucleolus 40,000 rRNA 50-70% II Nucleo- 40,000 hnRNA (precursor 20-40% plasm of mRNA) III Nucleo- 10,000 tRNA, 5s rRNA 10% plasm (small stable RNA’s) RNA polymerase: 6 sites of activity ⚫ DNA coding strand ⚫ RNA-DNA hybrid ⚫ DNA template strand ⚫ Unwinding point ⚫ RNA binding ⚫ Rewinding point  strand strand antisense / sense / coding / anticoding / plus (+) strand minus (-) strand DNA strand to the template in sequence with RNA transcribed (U instead of T)  sense strand 5’ ATGCCCGGTATGTTT 3’ 3’ 5’ antisense strand = template for transcription transcription 5’ 3’ primary transcript Three Stages of Transcription  Initiation of Transcription requires specific sequences = requires interaction of RNA pol with several proteins = or TFs  Promoter a sequence that “promotes” gene expression binding site of prokaryotic vs. eukaryotic promoters... strong vs. weak promoters...  Prokaryotic Promoter o by David Pribnow (Harvard University) o 10 bases before startpoint o Consensus sequence:  Prokaryotic Promoter upstream from Pribnow box consensus sequence:  Eukaryotic Promoters one for each type of RNA polymerase because there is a specific RNA polymerase for each type of Eukaryotic Promoters promoter for RNA pol II Eukaryotic Promoters promoter for RNA pol I * bipartite (core + UCE)  Eukaryotic Promoters promoter for RNA pol III *located within the gene it promotes Eukaryotic Promoters: general structure box or Goldberg/Hogness Box TATAWAW 19-27 bases before startpoint box ~70 bases before startpoint box ~40 bases before startpoint  Eukaryotic Promoters: general structure CAAT... GC.........TATAWAW....................... initiation site CAAT box Goldberg/ GC box Hogness box  Eukaryotic Promoters Roles 1. A-T rich region makes initial easier 2. of RNA polymerase relative to startpoint 3. Binding of auxillary proteins ( ) to facilitate RNA polymerase binding STRONG PROMOTER WEAK PROMOTER ⚫ same as consensus ⚫ significantly sequence from consensus sequence ⚫ RNA pol binds ⚫ RNA pol binds strongly ⚫ Initiation – ⚫ Initiation - more frequent frequent ⚫ RNA ⚫ More transcripts transcripts made made  Two Types of Promoter Mutation 1. mutation – causes deviation from consensus sequence eg. TATAAT consensus: TATAAT TAGCAT 2. mutation – causes a given sequence to become more/exactly similar to an established consensus sequence. Eg. TATGTT – lac operon TATATT  General Transcription Factors ⚫ protein factors ⚫ essential for transcription of any eukaryotic gene ⚫ sufficient to direct level of transcription from many core promoters ⚫ position RNA polymerase at the and send it on its way Assembly of Transcription Initiation Complex in Eukaryotes Gene-specific Transcription Factors proteins involved in interaction and activation of particular genes or groups of genes different sets in different cell types → different patterns of eg. – steroid hormone receptor – activates glucocorticoid response element – myogenic proteins – activates genes for muscle differentiation Gene-specific TFs General TFs RNA Polymerase ref: Becker’s World of the Cell  During transcription initiation … The RNA polymerase: 1. binds promoter 2. melts DNA 3. remains stationary 4. incorporates the 1st ribonucleotide -retains all 3 phosphate groups  Elongation of Transcription Nucleotides are added one at a time to the growing RNA chain. 5’ to 3’ wrt RNA product rapid: about 50 nucleotides/sec in E. coli Molecular Biology of the Cell. 3rd edition. Alberts B, Bray D, Lewis J, et al. 1994 During transcription elongation… Leading end of bubble unwinds while the tailing end is rejoined.  and ointercalates DNA oprevents movement of RNA pol oinhibits elongation  Termination of Transcription sequence signals RNA pol to stop. RNA transcript is. RNA polymerase.  Two Types of Termination 1. Rho- independent simple termination no need for rho factor ends in a stretch of rich in GC mRNA forms a  Two Types of Termination 2. Rho-dependent termination requires palindrome: less GC-rich no UUUUUU at end of RNA Rho binds to transcript at rut site and pursues RNA pol. Hairpin forms; RNA pol pauses; Rho catches up. Rho helicase releases transcript. 2. Rho dependent termination requires ATP