RNA polymerase binding sites

Steitz has suggested that DNA bending by CAP could contribute to activation of transcription by looping the DNA around CAP to provide for contacts between RNA polymerase and DNA upstream of the CAP-binding site. Such a model could explain how CAP can activate transcription from a variety of distances from the RNA polymerase-binding site since the size of the loop could vary. 

An amplification reaction that is used to amplify target RNA or denatured DNA is called the transcription-based amplification system (TAS). This technique involves using an enzyme called reverse transcriptase and a primer with sequence complementary to the sample target RNA molecule in order to synthesize a complementary DNA (cDNA) copy of the sample target RNA. After denaturation to separate the strands, another primer and additional reverse transcriptase are added to synthesize a double-stranded cDNA molecule. Since the first primer has also an RNA polymerase binding site, it can, in the presence of T7 RNA polymerase, amplify the double-stranded cDNA to produce 10 to 100 copies of RNA. The cycle of denaturation, synthesis of cDNA, and amplification to produce multiple RNA copies is repeated. With as few as four cycles, a 2- to 5-millionfold amplification of the original sample RNA target is possible. However, the time required to achieve a millionfold amplification is approximately 4 hours, which is the same amount of time required by PCR. The TAS requires, however, the addition of two enzymes at each cycle and, as such, can be cumbersome. 

FIGURE 1 Footp rint analysis of the RNA polymerase-binding site on a DNA fragment. Separate experiments are carried out in the presence (+) and absence (—) of the polymerase. 

RNA polymerase binding sites 5 -TATAATb Bacterial -10 or Pribnow promoter sequence... 

The GAL system shows both similarities and differences to typical bacterial regulatory systems. In both systems DNA-binding regulatory proteins play a major role. However, in the yeast system the binding site for the regulatory proteins is often located some distance upstream from the RNA polymerase-binding site. In yeast such distant sites required for activation are referred to as upstream activator sequences (UASs). 

The greatest difference between the regulatory systems in yeast and E. coli is that yeast regulatory proteins can bind at a long distance from the RNA polymerase-binding site and still be effective. 

The pWHA43 plasmid contains 3.9 kbp and replicates with the pMBl origin of replication. The plasmid was constructed with an insert of cDNA encoding met-prochymosin under the control of a tandem lac-trp promotor operator arrangement as described in (1). The prochymosin gene was inserted just upstream from the ampicillin resistance gene without a transcription terminator sequence between, but with the RNA polymerase binding site for amp also intact, so that presumably amp is transcribed from both the lac-trp and natural amp promotors. 

When glucose is depleted, the concentration of cAMP rises. The cAMP-CAP complex forms and binds to the CAP binding site just upstream of the RNA polymerase binding site in the lac promoter. At the same time, some lactose has entered the cell, has been converted to allolactose by (3-galactosidase, and is bound by the lac repressor so that it no longer binds to the lac operator. RNA polymerase now binds to the lac promoter even more effectively because of protein-protein interactions with the cAMP-CAP complex. The enzymes and permease of the lac operon are expressed fully consequently, lactose readily enters the cell and is efficiently metabolized. 

Some of the native B. subtilis chromosomal genes have also strong constitutive promoters such as the promoter of the rpsD gene, the P43 promoter [137], the lepA promoter [123], and the first RNA polymerase binding site of the tandem promoter complex of the veg gene [138]. The strength of the ve I promoter is comparable with that of the SPOl promoters. Interestingly, the transcription initiated from P j and its native partner, the second veg promoter, collide with each other and produce much less mRNA [139]. 

Le Grice, S.F. et al (1986) Separation and analysis of the RNA polymerase binding sites of a complex Bacillus... 

RNA polymerase binding sites. The vector is first linearized with a restriction enzyme such that transcription produces run-off transcripts, that is, transcripts derived from the insert sequence alone. The restriction enzyme is chosen such that transcription yields an RNA probe that is complementary (anti-sense) to the target mRNA. The probe transcribed from the opposite strand (sense probe) can be used as a negative control. The length of the probe is important. Probes of up to 1.5 kb are optimal. Longer probes penetrate the tissue less efficiently but can be partially degraded by alkaline hydrolysis to a more suitable size (2). 

The expression of proteins involved in Hg(II) detoxification is regulated by the MerR protein. The MerR protein is always bound as a dimer adjacent to the RNA polymerase binding site of the mer gene. In the absence of Hg, MerR holds the DNA in a conformation so that the RNA polymerase binding is blocked and transcription cannot occur. When the mercury binds to MerR, it changes the conformation of the MerR protein-DNA complex and allows RNA polymerase to bind and transcribe the mer operon, creating mRNA for the series of enzymes that carry out mercury resistance. 

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