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Transcription & Rna Processing Sibc504 SIBC504:SIBC504:SIBC504: TRANSCRIPTIONTRANSCRIPTIONTRANSCRIPTION &&& RNARNARNA PROCESSINGPROCESSINGPROCESSING Assistant Professor Dr. Chatchawan Srisawat TRANSCRIPTION: AN OVERVIEW Transcription: the synthesis of a single-stranded RNA from a double- stranded DNA template. • The first stage in the overall process of gene expression. • The most commonly-used step in regulating gene expression in organisms. Transcriptional control Switch gene on or off Phenotype TRANSCRIPTION: AN OVERVIEW Important features of transcription • RNA polymerase is the key enzyme: it requires DNA template, ribonucleotide precursors (ATP, GTP, CTP, and UTP) • It catalyzes the synthesis of RNA from ribonucleotide precursors using one of the two DNA strands as a template. Coding or sense strand Template or antisense strand • The sequence of the RNA transcript is the same as that of the coding strand (with U in place of T). TRANSCRIPTION: AN OVERVIEW Important features of transcription Coding or sense strand Template or antisense strand • Direction of synthesis occurs from the 5’- to the 3’-end of the RNA . TRANSCRIPTION: AN OVERVIEW Important features of transcription • Transcription process can be divided into three steps: 1. Initiation -1 +1 upstream downstream RNA polymerase transcription start site promoter A region of DNA involved in binding of RNA polymerase to initiate transcription; usually located upstream to the start site. • Binding of RNA polymease to specific DNA sequences called promoters. • Local unwinding of DNA and synthesizing the first few nucleotides of RNA (no primers required). TRANSCRIPTION: AN OVERVIEW Important features of transcription • Transcription process can be divided into three steps: 2. Elongation • Successive addition of ribonucleotides into the RNA transcript from 5’ to 3’ direction using DNA as a template. • DNA unwinding ahead of RNA polymerase and reannealing behind the enzyme. TRANSCRIPTION: AN OVERVIEW Important features of transcription • Transcription process can be divided into three steps: 3. Termination • Synthesis of the RNA transcript is stopped at the terminator sequence. RNA polymerase dissociates from the DNA and releases the RNA transcript. TRANSCRIPTION INITIATION Transcription initiation -1 +1 upstream downstream RNA polymerase transcription start site promoter • The first step of transcription process • Regulation of gene expression at transcriptional level are the most commonly used mode to determine which genes to be active or inactive. • Most of regulatory mechanisms of gene expression occur at the transcriptional initiation step. TRANSCRIPTION INITIATION IN PROKARYOTE • In prokaryotes, RNA polymerase is a multisubunit enzyme. RNA polymerase holoenzyme • Core enzyme • σ (sigma) factor alpha (α) -2subunits/enzyme - required for core protein assembly; may be involved in promoter binding beta (β) - involved in catalysis; chain initiation and elongation. beta’ (β’) - involved in template DNA binding. omega (ω) - promotes assembly. • Core enzyme can catalyze RNA synthesis and bind to DNA, but has no specificity;unable to start transcription at initiation sites alone. TRANSCRIPTION INITIATION IN PROKARYOTE • In prokaryotes, RNA polymerase is a multisubunit enzyme. RNA polymerase holoenzyme • Core enzyme • σ (sigma) factor • Responsible for binding of DNA at promoters. • Bring the core RNA polymerase enzyme to the initiation sites (promoters) to initiate transcription. • Required for initiation and released from the core enzyme at the start of transcription elongation. TRANSCRIPTION INITIATION IN PROKARYOTE σ factors and promoters • Many bacteria, including E. coli, produce a set of different σ factors. 70 32 28 38 54 Number denotes the molecular σ , σ , σ , σ , σ weight of σ factor. • σ70 is the most common σ factor found in E. coli. TRANSCRIPTION INITIATION IN PROKARYOTE σ factors and promoters upstream downstream promoter Promoter • A region of DNA involved in binding of RNA polymerase to initiate transcription; usually located upstream to the start site. • Different promoters contain conserved sequences, which can be specifically recognized and bound by different σ factors of RNA polymerase, thus facilitating assembly of the enzyme at the transcription initiation site . TRANSCRIPTION INITIATION IN PROKARYOTE σ70 factor and its promoter sequence •The σ70 promoter consists of a sequence of between 40-60 bp (region from around -55 to +20), which is bound by RNA polymerase. -10 sequence or Pribnow box consensus sequence -35 sequence G 70 TTGACA 16-18 bp TATAAT 5-8 bp CGT of σ promoter A Recognition region Important for DNA +1 interacting with σ factor unwinding Note - Different σ factors recognize specific promoters with different conserved sequences. TRANSCRIPTION INITIATION IN PROKARYOTE σ70 factor and its promoter sequence TRANSCRIPTION INITIATION IN PROKARYOTE Steps in transcription initiation complex formation • Promoter binding RNA polymerse core enzyme • The RNA polymerase core enzyme has a nonspecific affinity for DNA, making it difficult and very slow to search and bind to the promoter by itself. •With σ factor, the affinity for nonspecific sites on DNA is reduced by 20000-fold, while the affinity for promoters is enhanced by 100-fold. σ factor increases the specificity of the enzyme for promoter-binding sites. TRANSCRIPTION INITIATION IN PROKARYOTE Steps in transcription initiation complex formation • Promoter binding • At the promoter, the RNA polymerase recognizes the promoter sequences at -35 and -10 regions (via the interaction with σ factor) Formation of the initial complex of the RNA polymerase and the base- paired DNA at the promoter: a closed complex. TRANSCRIPTION INITIATION IN PROKARYOTE Steps in transcription initiation complex formation • DNA unwinding • Around 12 bp of the DNA (from -9 to +3) is unwound by the polymerase, forming an open comlex. • -10 sequence region (AT-rich) is important for DNA unwinding TRANSCRIPTION INITIATION IN PROKARYOTE Steps in transcription initiation complex formation • RNA chain initiation -35 sequence -10 sequence or Pribnow box G TTGACA 16-18 bp TATAAT 5-8 bp CGT A +1 • RNA polymerase starts synthesizing a short RNA chain without the movement of the enzyme from the promoter. • The synthesis does not require a primer. A short RNA chain of 9 nt in length is generated; the first base is usually started with G or A. • The process is ineffective. It may abort and then restart until initiation succeeds. TRANSCRIPTION INITIATION IN PROKARYOTE Steps in transcription initiation complex formation • RNA chain initiation • When initiation succeeds, the enzyme releases the σ factor (which is not required for elongation) and forms a ternary complex of polymerase-DNA-RNA transcript. • Transcription elongation begins as the enzyme moves along the DNA to synthesize RNA transcript. TRANSCRIPTION INITIATION IN PROKARYOTE Alternative sigma factors • Sigma factors confer promoter specificity to the core RNA polymerase. • The presence of alternate sigma factors provides the cell with a mechanism for turning on and off entire families of genes under different circumstances. Note- Deviation of a promoter sequence from the consensus may affect the promoter strength and efficiency of transcription TRANSCRIPTION INITIATION IN EUKARYOTE • In eukaryotes, there are many kinds of RNAs with different functions. • There are also three different kinds of RNA polymerases (Pol I, II, and III) to transcribe these RNAs in eukaryotic cells. TRANSCRIPTION INITIATION IN EUKARYOTE Eukaryotes have 3 types of RNA polymerases responsible for transcription of nuclear genes • RNA polymerase I – transcribes rRNA • RNA polymerase II - transcribes mRNA (all protein-coding RNA) • RNA polymerase III - transcribes tRNA, 5S RNA, snRNA, 7SL RNA Distinguished by sensitivity to α-amanitin • In addition, there are organelle-specific RNA polymerases (i.e., mitochondria and chloroplast) to transcribe genes in the organelles. TRANSCRIPTION INITIATION IN EUKARYOTE Comparison of structures of eukaryotic and prokaryotic RNA polymerases • Eukaryotic RNA polymerase (Pol I, II, and III) are multimeric enzymes and some of the subunits are conserved, showing similarity to the prokaryotic counterparts. Note- the carboxy terminus (CTD) of β’ like subunit of RNA pol II contains repeats of seven amino acids – YSPTSPS - (up to 50 repeats), which can be phosphorylated during transcription. TRANSCRIPTION INITIATION IN EUKARYOTE Typical RNA polymerase II core promoter Core promoter: the minimal DNA region at which RNA polymerase II can bind and initiate basal transcription of the gene. 1. TATA box - located approximately 25- 30 bp upstream of the start site A A - the conserved sequence: 5’ TATA T AT 3’ 2. Initiator element (Inr) - usually located at start site - the conserved sequence: 5’ Py2CAPy5 3’ TRANSCRIPTION INITIATION IN EUKARYOTE Transcription initiation of RNA polymerase II • Like the core enzyme of RNA polymerase in prokaryotes, the RNA polymerases in eukaryotes also cannot bind to the promoter by itself. • General or basal transcription factors are required for RNA pol II to initiate transcription at the promoter. TRANSCRIPTION INITIATION IN EUKARYOTE Transcription initiation of RNA polymerase II General or basal transcription factors • Not subunits of the RNA polymerase II. • Required for RNA polymerase to bind avidly and specifically to promoters. • General transcription factors for RNA polymerase II are called TFIIx, where x = A, B, D,... • Can have multiple subunits. TRANSCRIPTION INITIATION IN EUKARYOTE
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