RNA Transcription PDF

Anti-sigma factors Anti-sigma factors bind sigma factors and inhibit their functions. Example: an anti-sigma factor can control general stress response in M. extorquens. Phosphorylation of PhyR is induced by stress which results in release of anti-sigma factor (NepR) from the extracytopl...

Anti-sigma factors Anti-sigma factors bind sigma factors and inhibit their functions. Example: an anti-sigma factor can control general stress response in M. extorquens. Phosphorylation of PhyR is induced by stress which results in release of anti-sigma factor (NepR) from the extracytoplasmic function (ECF) sigma factor σEcfG1. Thus releasing σEcfG1 and allowing σEcfG1 to associate with RNA polymerase to transcribe stress genes. Francez-Charlot et al. PNAS 2009  Transcription start and elongation 1. Formation of the closed complex: Sigma factor The RNA polymerase bound to the promoter region is referred to as the "closed" complex. DNA is still Closed double-stranded. complex 2. Formation of the open complex: The DNA duplex unwinds at the Core enzyme promoter region catalyzed by the sigma factor to form the “open” complex. There is no requirement for a helicase or a nucleotide triphosphate such as ATP. A small set Open of aromatic residues within sigma complex factor help to open the DNA at the A/T-rich -10 region as it "breathes" or transiently melts.  Transcription start and chain elongation 3. Binding of initiating ribonucleotides Transcription is usually initiated at the start site (+1) with a nucleotide Binding of triphosphate. The newly synthesized initiating RNA therefore has a triphosphate at ribonucleotides its 5' end. 4. Elongation The sigma subunit dissociates from the polymerase and formation of a DNA opening of 18 bases long, called transcription bubble, in which the elongating RNA forms an Elongation RNA/DNA hybrid with the template (transcription DNA strand. The RNA/DNA hybrid bubble) helps keep the RNA polymerase attached to the DNA.  Features of chain elongation Transcription can occur in either strand of DNA depending on the genes involved. Multiple RNA polymerases could transcribe the same gene at the same time. Each “branch” is an RNA polymerase  Polycistronic mRNA Polycistronic mRNA: one single mRNA molecule contains multiple genes. They are transcribed from the same promoter upstream of a cluster of genes which are usually of related functions – this cluster is called an operon. Related biological function Ribosomal RNA is polycistronic Figure 4.22 Coupled transcription and translation in bacteria In bacteria, translation and transcription are coupled. What happens is that ribosomes attach to the 5' end of the mRNA even before the message is completely transcribed. Then they move along the mRNA toward the 3' end as transcription continues. Because translation is coupled to transcription, proteins can begin to be made even before the mRNA is completed, thus shortening the delay between the onset of transcription and the appearance of the protein. Coupled transcription and translation can not take place in eukaryotes, since transcription and translation occur in separate cell compartments (i.e. the nucleus and cytoplasm, respectively).  Transcription termination When the RNA polymerase hits a DNA sequence called transcription terminator, it will fall off from the template and transcription is halted. Termination include factor-independent and Rho-dependent types. “Factor” here means a protein factor. Factor-independent termination (also called intrinsic termination and Rho-independent termination ): - A common termination sequence is an inverted repeat (high G+C) with a central non-repeating segment forming a hairpin structure by intramolecular base-pairing. - Following the inverted repeat sequence, there is a run of U. These sequences/structures destabilize RNA/DNA binding which leads to RNA dissociation from DNA.  Rho-dependent termination There are three termination factors in E. coli. They are Rho, Tau and NusA. The best studied is Rho, which is an RNA-dependent ATPase and an ATP- dependent RNA/DNA helicase. Terminator sequences are not closely related Polymerase pausing and are not always easy to recognize. Rho binds to rut (rho utilization sites) in the RNA directly behind the RNA polymerase. rut When rut sequences are not associated with Rho ribosomes, Rho hexamer will form on rut and then acts as an ATP-dependent RNA helicase, rapidly moving along the transcript in the 5‘ to 3' direction, pulling apart the RNA/DNA hybrid duplex, which results in the RNA polymerase disengagement (when RNA www.slideshare.net polymerase is paused at the terminator sequence).  Distinctions between bacterial and eukaryotic RNA transcriptions Bacterial transcription Eukaryotic transcription One RNA polymerase Three (or more) RNA polymerases RNA polymerase has 5 subunits RNA polymerase has more than and needs sigma factors. 10 subunits and use transcription factors mRNA has 5’ triphosphate end mRNA has 5’ cap modification Multiple genes in one mRNA One gene in one mRNA (polycistronic mRNA) (monocistronic) Coupled transcription and Transcription in nucleus and translation translation in cytoplasm mRNA has no introns mRNA have many introns and will be spliced