RNA polymerase initiation

DNA-Dependent RNA Polymerase Initiates Transcription at a Distinct Site, the Promoter... 

FIGURE 28-3 Consensus sequence for promoters that regulate expression of the E. coli heat-shock genes. This system responds to temperature increases as well as some other environmental stresses, resulting in the induction of a set of proteins. Binding of RNA polymerase to heat-shock promoters is mediated by a specialized a subunit of the polymerase, cr32, which replaces cr70 in the RNA polymerase initiation complex. 

RNA polymerase activity is discovered in crude extracts of cells derived from an exotic fungus. The RNA polymerase initiates transcription only from a single, highly specialized promoter. As the polymerase is purified its activity declines, and the purified enzyme is completely inactive unless crude extract is added to the reaction mixture. Suggest an explanation for these observations. 

Correct answer = A. The addition of glucose causes cyclic AMP production to decrease. In the absence of cyclic AMP, the CAP protein cannot remain bound to its DNA binding site. An empty CAP binding site is not able to help RNA polymerase initiate transcription, so the rate of transcription decreases. Lower mRNA produc tion results in decreased P-galactosidase syn thesis. Because lactose is still present, the inducer (allolactose) remains bound to the repressor, which continues to be unable to bind to the operator. 

RNA polymerase initiates de novo, and does not require a primer, unlike DNA polymerase. Finally, RNA polymerase does not perform any proofreading activity, unlike DNA polymerases, which generally have a 3 to 5 exonuclease activity to remove mis-incorporated nucleotides. 

Liu, C., and Martin, C. T. (2002). Promoter clearance by T7 RNA polymerase. Initial bubble collapse and transcript dissociation monitored by base analog fluorescence. J. Biol. Chem. 277, 2725-2731. 

Bouche, J.-P., Zechel, K., Komberg, A. DNA G gene product, a rifampicin resistant RNA polymerase, initiates the conversion of a single-stranded coliphage DNA to its duplex replicative form. J. Biol. Chem. 250, 5995 (1975)... 

One of the first metalloregulatory proteins to be characterized extensively is the prokaryotic MerR transcription factor (1, 6, 7), which acts either as a repressor (apo-protein) or an activator (holo-protein) of the mer operon encoding mercury resistance proteins (Fig. Ic). The —35 and —10 sequence elements of the mer promoter, binding sites for the RNA polymerase initiation complex, are separated by an unusually long distance that results in poor constitutive transcription. Apo-MerR binds to the DNA between these sequences and bends the DNA, which results in a slight increase in repression on the suboptimal promoter. It also recruits the RNA polymerase to the transcription start site where it waits in a stalled complex. Upon binding of... 

G.M. Cheetham and T.A. Steitz. 1999. Structure of a transcribing T7 RNA polymerase initiation complex Science 286 2305-2309. (PubMed)... 

Cheetham, G. M., and Steitz, T. A. 1 999. Structure of a transcribing T7 RNA polymerase initiation complex. Science 286 2305-2309. Ebright, R. H. 2000. RNA polymerase Structural similarities between bacterial RNA polymerase and eukaryotic RNA polymerase II. j. Mol. Biol. 304 687—698. 

See also Eukaryotic Transcription (from Chapter 28) RNA Polymerases, Initiation and Elongation, Promoter Organization, Factor-Independent Termination of Transcription, Factor-Dependent Termination of Transcription Transcription Regulation in Phage, trp Operon Regulation... 

RNA polymerase initiates mRNA synthesis, almost altra) with a purine... 

The current concept of a classic operon is largely derived from studies of the lac operon of E. coli and includes a promoter at the beginning which is considered to be the site at which the DNA-dependent RNA polymerase initiates transcription [151,152]. The promoter is followed by a closely associated operator serving as the site of attachment for repressor molecules which can prevent or impede transcription [153]. The structural genes are next in the sequence, and in the case of the tryptophan operon they code for the peptides which make up the tryptophan biosynthetic enzymes. Presumably there is an element at the end of the operon which acts as a signal to terminate transcription. This section will describe information derived primarily from work with E. coli and S. typhimurium. A number of general reviews and discussions of regulation have been published recently [4-6,105,154]. 

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