Chapter Eleven Transcription of the Genetic Code: The Biosynthesis of RNA Chapter 11 Transcription • Overview of Transcription • synthesized on a DNA template, catalyzed by DNA- dependent RNA polymerase • ATP, GTP, CTP, and UTP are required, as is Mg2+ • no RNA primer is required • the DNA base sequence contains signals for initiation and termination of RNA synthesis; the enzyme binds to and moves along the DNA template in the 3’ -> 5’ direction – the RNA chain is synthesized in the 5’ -> 3’ direction • the DNA template is unchanged Transcription in Prokaryotes • E. coli RNA Polymerase: – molecular weight about 500,000 – four different types of subunits: , and s , ’, – the core enzyme is ’2 – the holoenzyme is ’s 2 – the role of the s subunit is recognition of the promoter locus; locus the s subunit is released after transcription begins – of the two DNA strands, the one that serves as the template for RNA synthesis is called the template strand or antisense strand; strand the other is called the coding (or nontemplate) strand or sense strand – the holoenzyme binds to and transcribes only the template strand The Basics of Transcription Promoter Sequence • Simplest of organisms contain a lot of DNA that is not transcribed • RNA polymerase needs to know which strand is template strand, which part to transcribe, and where first nucleotide of gene to be transcribed is • Promoters-DNA sequence that provide direction for RNA polymerase Promoter Sequence How does RNA polymerase know where to begin transcription? • Polymerase moves along template strand from 3’-5’ and RNA is formed from 5’-3’ • Binding site for polymerase is upstream of start of transcription – away from 5’ of coding strand • Promoter sequence is based on coding strand and RNA polymerase is binding to template strand How does RNA polymerase know where to begin transcription? • Promoter are upstream towards 5’ of coding and 3’ template strand • The first base to be incorporated is at position + 1 = transcription start site • All nucleotides upstream from this start site are given –ve numbers • The first promoter element is about 10 bases upstream is – 10 region or pribnow box How does RNA polymerase know where to begin transcription? • After PB 16-18 bases are variable • Next promoter element is about 35 bases upstream of TSS = -35 region or -35 element • Area from -35 element to TSS = core promoter • Upstream of core element is UP element = enhances binding of RNA polymerase • Region from UP element to TSS = extended promoter • BASE SEQUENCE of promoter is A and T Chain Initiation • First phase of transcription is initiation • Initiation begins when RNA polymerase binds to promoter and forms closed complex • After this, DNA unwinds at promoter to form open complex, which is required for chain initiation Initiation and Elongation Transcription Chain Elongation (Cont’d) Chain Elongation • After strands separated, transcription bubble of ~17 bp moves down the DNA sequence to be transcribed • RNA polymerase catalyzes formation of phosphodiester bonds between the incorp. ribonucleotides • Topoisomerases relax supercoils in front of and behind transcription bubble Chain Termination • Two types of termination mechanisms: • intrinsic termination- controlled by specific sequences, termination sites • Termination sites characterized by two inverted repeats Chain Termination • Other type of termination involves rho () protein • Rho-dependent termination sequences cause hairpin loop to form Transcription Regulation in Prokaryotes • In prokaryotes, transcription regulated by: • alternative s factors – enhancers – operons – transcription attenuation Alternative s factors • Viruses and bacteria exert control over which genes are expressed by producing different s-subunits that direct the RNA polymerase to different genes. Enhancers • Certain genes include sequences upstream of extended promoter region • These genes for ribosomal production have 3 upstream sites, Fis sites • Class of DNA sequences that do this are called enhancers • Bound by proteins called transcription factors Elements of a Bacterial Promoter Operon • Operon: a group of operator, promoter, and structural genes that codes for proteins – the control sites, promoter, and operator genes are physically adjacent to the structural gene in the DNA – the regulatory gene can be quite far from the operon – operons are usually not transcribed all the time Example of Operon system • b-Galactosidase, an inducible protein – coded for by a structural gene, lacZ – structural gene lacY codes for lactose permease – structural gene lacA codes for transacetylase – expression of these three structural genes is controlled by the regulatory gene lacI that codes for a repressor Transcription in Eukaryotes is complex • Three RNA polymerases are known - each transcribes a different set of genes and recognizes a different set of promoters: • RNA Polymerase I- found in the nucleolus and synthesizes precursors of most rRNAs • RNA Polymerase II- found in the nucleoplasm and synthesizes mRNA precursors • RNA Polymerase III- found in the nucleoplasm and synthesizes tRNAs, other RNA molecules involved in mRNA processing and protein transport RNA Polymerase II • Most studied in the polymerases • Consists of 12 subunits • RPB- RNA Polymerase B How does Pol II Recognize the Correct DNA? • Four elements of the Pol II promoter . Pol II promoters • Variety of upstream elements – activators and silencers - GC box (-40) – Consensus sequence – GGGCGG - CAAT box (extending to – 110) – Consensus sequence - GGCCAATCT Pol II promoters • Second element found at -25 = TATA box has consensus sequence of TATAA (T/A) • Transcription start site at position + 1 surrounded by a sequence called initiator element (Inr) • Inititator and TATA box = core promoter • Fourth element – downstream regulator - rare Initiation of Transcription • Transcription factor - Any protein regulator of transcription that is not a subunit of Pol II • Initiation begins by forming the preinitiation complex - Transcription control is based here Transcription Order of Events • The phosphorylated Pol II synthesizes RNA • Leaves the promoter region behind • GTFs are left at the promoter or dissociate from Pol II Elongation and Termination • Elongation is controlled by: – pause sites - where RNA Pol will hesitate – positive transcription elongation factor (P-TEF) and negative transcription elongation factor (N- TEF) • Termination – begins by stopping RNA Pol; the eukaryotic consensus sequence for termination is AAUAAA Gene Regulation • Enhancers and silencers- regulatory sequences that augment or diminish transcription, respectively • DNA looping brings enhancers into contact with transcription factors and polymerase Response elements • Response elements are enhancers that respond to certain metabolic factors • heat shock element (HSE) • glucocorticoid response element (GRE) • metal response element (MRE) • cyclic-AMP response element (CRE) Structural Motifs in DNA-Binding Proteins • Most proteins that activate or inhibit RNA Pol II have two functional domains: – DNA-binding domain – transcription-activation domain • DNA-Binding domains have domains that are either: • Helix-Turn-Helix (HTH) • Zinc fingers • Basic-region leucine zipper Helix-Turn-Helix Motif Hydrogen bonding between amino acids and DNA Zinc Finger Motif • Motif contains 2 cysteines and 2 His – after every 12 amino acids • Zinc binds to the repeats Basic Region Leucine Zipper Motif • Many transcription factors contain this motif - CREB • Half of the protein composed of basic region of conserved Lys, Arg, and His • Half contains series of Leu Post Transcriptional RNA Modification • tRNA, rRNA, and mRNA are all modified after transcription to give the functional form – the initial size of the RNA transcript is greater than the final size because of the leader sequences at the 5’ end and the trailer sequences at the 3’ end • Modifications – trimming of leader and trailer sequences – addition of terminal sequences (after transcription) – modification of specific bases (particularly in tRNA) Modification of tRNA • Transfer RNA- – trimming, addition of terminal sequences, and base modification - take place – methylation and substitution of sulfur for oxygen are the two most usual types of base modification Modification of rRNA • Ribosomal RNA – processing of rRNA - methylation and trimming to the proper size Modification of mRNA • Capping of the 5’ end with an N-methylated guanine • A polyadenylate “tail” that is usually100-200 nucleotides long, is added to the 3’ end before the mRNA leaves the nucleus Organization of Split Genes in Eukaryotes Modification of mRNA – Eukaryote genes frequently contain intervening base sequences that do not appear in the final mRNA of that gene product – Expressed DNA sequences are called exons – Intervening DNA sequences that are not expressed are called introns – These genes are often referred to as split genes The Splicing Reaction • Exons are separated by intervening intron • When the exons are spliced together - lariat forms in the intron Ribozymes • The first ribozymes discovered included those that catalyze their own self-splicing • ribozymes have been discovered that are involved in protein synthesis • Group I and II • This project is funded by a grant awarded under the President’s Community Based Job Training Grant as implemented by the U.S. Department of Labor’s Employment and Training Administration (CB-15-162-06-60). 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