Presentation on theme: "Transcription Overview of Transcription"— Presentation transcript:
1 Chapter Eleven Transcription of the Genetic Code: The Biosynthesis of RNA
2 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’ directionthe RNA chain is synthesized in the 5’ -> 3’ direction• the DNA template is unchanged
3 Transcription in Prokaryotes E. coli RNA Polymerase:molecular weight about 500,000four different types of subunits: , , ’, and sthe core enzyme is 2’the holoenzyme is 2’sthe role of the s subunit is recognition of the promoter locus; the s subunit is released after transcription beginsof the two DNA strands, the one that serves as the template for RNA synthesis is called the template strand or antisense strand; the other is called the coding (or nontemplate) strand or sense strandthe holoenzyme binds to and transcribes only the template strand
5 Promoter SequenceSimplest of organisms contain a lot of DNA that is not transcribedRNA polymerase needs to know which strand is template strand, which part to transcribe, and where first nucleotide of gene to be transcribed isPromoters-DNA sequence that provide direction for RNA polymerase
7 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 strandPromoter sequence is based on coding strand and RNA polymerase is binding to template strand
8 How does RNA polymerase know where to begin transcription? Promoter are upstream towards 5’ of coding and 3’ template strandThe first base to be incorporated is at position + 1 = transcription start siteAll nucleotides upstream from this start site are given –ve numbersThe first promoter element is about 10 bases upstream is – 10 region or pribnow box
9 How does RNA polymerase know where to begin transcription? After PB bases are variableNext promoter element is about 35 bases upstream of TSS = -35 region or -35 elementArea from -35 element to TSS = core promoterUpstream of core element is UP element = enhances binding of RNA polymeraseRegion from UP element to TSS = extended promoterBASE SEQUENCE of promoter is A and T
10 Chain Initiation First phase of transcription is initiation Initiation begins when RNA polymerase binds to promoter and forms closed complexAfter this, DNA unwinds at promoter to form open complex, which is required for chain initiation
13 Chain ElongationAfter strands separated, transcription bubble of ~17 bp moves down the DNA sequence to be transcribedRNA polymerase catalyzes formation of phosphodiester bonds between the incorp. ribonucleotidesTopoisomerases relax supercoils in front of and behind transcription bubble
14 Chain Termination Two types of termination mechanisms: • intrinsic termination- controlled by specific sequences, termination sitesTermination sites characterized by two inverted repeatsThe inverted repeats are sequences of bases that are complementary such that they can loop back on themselves. Dna codes a series of uracils. The A-U base pairs between template strand forming hairpin loop. These are weakly bonded compared with gc pairs and rna dissociates from transcription bubble ending transcription
15 Chain Termination Other type of termination involves rho () protein Rho-dependent termination sequences cause hairpin loop to formRho sequences form hair loop and rho protein will stall polymerase from proceeding
16 Transcription Regulation in Prokaryotes In prokaryotes, transcription regulated by:• alternative s factorsenhancersoperonstranscription attenuation
17 Alternative s factorsViruses and bacteria exert control over which genes are expressed by producing different s-subunits that direct the RNA polymerase to different genes.
18 EnhancersCertain genes include sequences upstream of extended promoter regionThese genes for ribosomal production have 3 upstream sites, Fis sitesClass of DNA sequences that do this are called enhancersBound by proteins called transcription factors
20 OperonOperon: a group of operator, promoter, and structural genes that codes for proteinsthe control sites, promoter, and operator genes are physically adjacent to the structural gene in the DNAthe regulatory gene can be quite far from the operonoperons are usually not transcribed all the time
21 Example of Operon system b-Galactosidase, an inducible proteincoded for by a structural gene, lacZstructural gene lacY codes for lactose permeasestructural gene lacA codes for transacetylaseexpression of these three structural genes is controlled by the regulatory gene lacI that codes for a repressor
22 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 transportProkaryotes have single RNA polymerase responsible for three kinds of prokayotic RNA - m RNA, t RNA and r RNA. THE polymerase switch sigma factors to interact with different promoters but core polymerase stays same. All three eukaryotic RNA are large and consist of 10 or more subunits. Their overall structure differs but have two larger subunits that share homology with ß and ß1 of prokaryotic RNA polymerase. There are no sigma subunits to direct polymerases to promoters.
23 RNA Polymerase II Most studied in the polymerases Consists of 12 subunitsRPB- RNA Polymerase BRPB1-RPB12. I, II, III also known as A, B and C
24 How does Pol II Recognize the Correct DNA? Four elements of the Pol II promoter .
25 Pol II promotersVariety of upstream elements – activators and silencersGC box (-40) – Consensus sequence – GGGCGGCAAT box (extending to – 110) – Consensus sequence - GGCCAATCT
26 Pol II promotersSecond 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 promoterFourth element – downstream regulator - rareALL four are not present in all eukaryotes. Core promoter is most consistent in most species and genes
27 Initiation of Transcription Transcription factor -Any protein regulator of transcription that is not a subunit of Pol IIInitiation begins by forming the preinitiation complex - Transcription control is based hereGeneral transcription factors
28 Transcription Order of Events The phosphorylated Pol II synthesizes RNALeaves the promoter region behindGTFs are left at the promoter or dissociate from Pol IILess is known about eukaryotes than prokaryotes. Pol II gets dephosphorylated and recycles back
29 Elongation and Termination Elongation is controlled by:pause sites - where RNA Pol will hesitatepositive transcription elongation factor (P-TEF) and negative transcription elongation factor (N-TEF)Terminationbegins by stopping RNA Pol; the eukaryotic consensus sequence for termination is AAUAAAElongation factors increase the productive form and decrease abortive form
30 Gene RegulationEnhancers and silencers- regulatory sequences that augment or diminish transcription, respectivelyDNA looping brings enhancers into contact with transcription factors and polymerase
31 Response elementsResponse 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)
32 Structural Motifs in DNA-Binding Proteins Most proteins that activate or inhibit RNA Pol II have two functional domains:DNA-binding domaintranscription-activation domainDNA-Binding domains have domains that are either:• Helix-Turn-Helix (HTH)• Zinc fingers• Basic-region leucine zipperInteract through hydrogen bonding, electrostatic attractions and hydrophobic interactions
33 Helix-Turn-Helix Motif Hydrogen bonding between amino acids and DNA
34 Zinc Finger MotifMotif contains 2 cysteines and 2 His – after every 12 amino acidsZinc binds to the repeats
35 Basic Region Leucine Zipper Motif Many transcription factors contain this motif - CREBHalf of the protein composed of basic region of conserved Lys, Arg, and HisHalf contains series of Leu
36 Post Transcriptional RNA Modification tRNA, rRNA, and mRNA are all modified after transcription to give the functional formthe 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’ endModificationstrimming of leader and trailer sequencesaddition of terminal sequences (after transcription)modification of specific bases (particularly in tRNA)the types of processing in prokaryotes can differ greatly from that in eukaryotes, especially for mRNA
37 Modification of tRNA Transfer RNA- trimming, addition of terminal sequences, and base modification - take placemethylation and substitution of sulfur for oxygen are the two most usual types of base modification
38 Modification of rRNA Ribosomal RNA processing of rRNA - methylation and trimming to the proper size
39 Modification of mRNACapping of the 5’ end with an N-methylated guanineA polyadenylate “tail” that is usually nucleotides long, is added to the 3’ end before the mRNA leaves the nucleusThis tail protects the mRNA from nucleases and phosphatases
41 Modification of mRNAEukaryote genes frequently contain intervening base sequences that do not appear in the final mRNA of that gene productExpressed DNA sequences are called exonsIntervening DNA sequences that are not expressed are called intronsThese genes are often referred to as split genes
42 The Splicing Reaction Exons are separated by intervening intron When the exons are spliced together - lariat forms in the intron
43 RibozymesThe first ribozymes discovered included those that catalyze their own self-splicingribozymes have been discovered that are involved in protein synthesisGroup I and II
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