Transcript Slide 1
Transcription 5----3 direction RNA polymerase consensus sequence Initiation Elongation Termination α2ββ’σ σ = Initiation factor Promotor -35 = TTGACA -10 = TATAAT (Pribnow box) ρ dependent, independent Stem& Loop ρ Factor Structural similarity between a bacterial RNA polymerase and a eucaryotic RNA polymerase II. Regions of the two RNA polymerases that have similar structures are indicated in green. The eucaryotic polymerase is larger than the bacterial enzyme (12 subunits instead of 5), and some of the additional regions are shown in gray. The blue spheres represent Zn atoms that serve as structural components of the polymerases, and the red sphere represents the Mg atom present at the active site, where polymerization takes place. The RNA polymerases in all modern-day cells (bacteria, archaea, and eucaryotes) are closely related, indicating that the basic features of the enzyme were in place before the divergence of the three major branches of life Transcription Bubble. A schematic representation of a transcription bubble in the elongation of an RNA transcript. Duplex DNA is unwound at the forward end of RNA polymerase and rewound at its rear end. The RNA-DNA hybrid rotates during elongation. The structure of a bacterial RNA polymerase. Two depictions of the three-dimensional structure of a bacterial RNA polymerase, with the DNA and RNA modeled in. This RNA polymerase is formed from four different subunits, indicated by different colors (right). The DNA strand used as a template is red, and the non-template strand is yellow. The rudder wedges apart the DNA-RNA hybrid as the polymerase moves. For simplicity only the polypeptide backbone of the rudder is shown in the righthand figure, and the DNA exiting from the polymerase has been omitted. Because the RNA polymerase is depicted in the elongation mode, the σ factor is absent Schematic representation of the major form of E. coli RNA polymerase bound to DNA. By convention, the transcription-initiation site is generally numbered +1. Base pairs extending in the direction of transcription are said to be downstream of the start site; those extending in the opposite direction are upstream. The σ70 subunit binds to specific sequences near the −10 and −35 positions in the promoter. The α subunits lie close to the DNA in the upstream direction. The β and β′ subunits associate with the start site Alternative Promoter Sequences. A comparison of the consensus sequences of standard promoters, heat-shock promoters, and nitrogen-starvation promoters of E. coli. These promoters are recognized by σ70, σ32, and σ54, respectively. Members of the s70 family of sigma factors have 4 conserved regions Region 1 masks the DNA binding activities that are present in regions 2 and 4, which become unmasked when the sigma factor binds to the core RNA polymerase Region 2 Subregion 2.3 and 2.4 form a DNA binding activity that recognizes the -10 promoter motif, region 2 also interacts with core enzyme components Region 4: a DNA binding activity that recognizes the -35 promoter motif subunit size aa size (Kd) gene function alpha () 329 36511 rpoA required for assembly of the enzyme; interacts with some regulatory proteins; also involved in catalysis beta (b) 1342 150616 rpoB involved in catalysis: chain initiation and elongation beta' (b') 1407 155159 rpoC binds to the DNA template sigma (s) 613 70263 rpoD directs enzyme to the promoter rpoZ required to restore denatured RNA polymerase in vitro to its fully functional form omega (w) 91 10237 sigma factor gene function s70 rpoD principal sigma factor s54 rpoN (ntrA, glnF) nitrogen-regulated gene transcription s32 rpoH heat-shock gene transcription sS rpoS gene expression in stationary phase cells sF rpoF expression of flagellar operons sE rpoE involved in heat shock and oxidative stress responses; regulates expression of extracytoplasmic proteins sFecI fecI regulates the fec genes for iron dicitrate transport Sigma Factor Promoters Recognized Promoter Consensus −35 Region −10 Region σ70 Most genes TTGACAT TATAAT σ32 Genes induced by heat shock TCTCNCCCTTG AA CCCCATN TA σ28 Genes for motility and chemotaxis CTAAA CCGATAT σ38 Genes for stationary phase and stress response σ54 Genes for nitrogen metabolism and other functions ? ? −24 Region −12 Region CTGGNA TTGCA Structure of the σ Subunit. The structure of a fragment from the E. coli subunit σ70 reveals the position of an α helix on the protein surface; this helix plays an important role in binding to the -10 TATAAT sequence. RNA-DNA Hybrid Separation. A structure within RNA polymerase forces the separation of the RNA-DNA hybrid, allowing the DNA strand to exit in one direction and the RNA product to exit in another Mechanism For the Termination of Transcription by ρ Protein. This protein is an ATP-dependent helicase that binds the nascent RNA chain and pulls it away from RNA polymerase and the DNA template. Rho factor Rho-dependent termination. Rho is a helicase that follows the RNA polymerase along the transcript. When the polymerase stalls at a hairpin, Rho catches up and breaks the RNA-DNA base pairs, releasing the transcript. Note that the diagram is schematic and does not reflect the relative sizes of Rho and the RNA polymerase. Termination at an intrinsic terminator. The presence of an inverted palindrome in the DNA sequence results in formation of a hairpin loop in the transcript Termination Signal. A termination signal found at the 3′ end of an mRNA transcript consists of a series of bases that form a stable stem-loop structure and a series of U residues Rho Independent Transcription Termination Rho Dependent Transcription Termination