Promotor sequence

Random oligonucleotide mutagenesis was first applied to promotor sequences that regulate the production of enzymes in cells [21] and was the first method used to alter systematically the functions of enzymes by directed evolution [22], Based on our experience, we will focus on this approach and emphasize recent applications of this methodology to enzymes involved in DNA repair and synthesis, including DNA polymerase enzymes, thymidylate synthase, thymidine kinase, and 06-alkylguanine-DNA alkyltransferase. 

RNase P, which removes the 5 leader from tRNA transcripts, contains an RNA moiety in bacteria, eucarya, and archaea. The enzyme from Haloferax volcanii has been purified and the 345-nucleotide RNA portion used to generate a probe for cloning of the corresponding gene [123]. The sequence can be folded into a structure similar to that of bacterial RNase P RNAs. SI nuclease and primer extension localize the 5 end of the transcript adjacent to four potential archaeal promotor sequences. An in vitro transcript corresponding to the native RNA plus twenty 5 and nine 3 flanking nucleotides did not by itself exhibit RNase P activity (as bacterial RNase P RNAs do), under a variety of conditions. It was, however, able to reconstitute an active enzyme in combination with the protein moiety from Bacillus subtilis. This indicates that in Haloferax volcanii the RNase P RNA is the catalytic part of the enzyme but it may require some structural help [123]. 

Norinder, U. (1994). Theoretical Descriptors of Nucleic Acid Bases. Application to DNA Promotor Sequences. QuantStruct.-ActRelat., 13,295-301. 

Prestridge, D. S. (1995) Predicting Pol II promotor sequences using transcription factor binding sites. J Mol Biol 249, 923-932. 

Transcription initiation in procaryotes is controlled via promotors and regulatory DNA sequences located near the promotor. The role of the promotor is to provide a defined association site for the RNA polymerase and to correctly orient it. The binding of the RNA polymerase to its promotor is controlled by the sigma factor, a component of the RNA polymerase holoenzyme. The sigma factor selects which genes are to be transcribed by specifically recognizing the promotor sequence and structure and by allowing the RNA polymerase to form a transcription-competent complex at the transcription start site. 

An important aspect of dependent promotors is the fact that the holoenzyme is capable of initiating a weak transcription even without accessory proteins. In this way, the regulatory proteins is possible. In this case, the extent of transcription depends on the affinity of the holoenzyme for the promotor and thus depends indirectly on the promotor sequence. 

Repressors turn off transcription by, for example, competing with the holoenzyme for binding to the promotor. RNA polymerases and repressors compete if the repressorbinding site overlaps with the promotor sequence. 

Promotor sequence (Section 28.4) A short sequence on DNA located upstream of the transcription start site and recognized by RNA polymerase. 

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