[Structural-functional analysis of the promoter region of Escherichia coli udp gene]. / Strukturno-funktsional'nyi analiz promotornoi oblasti gena udp Escherichia coli.

Effect of mutations in the -10 and -35 regions of the udp gene promoter on the nature of its regulation by CytR and CRP proteins was studied. In studies of expression of mutant promoters, competition between RNA polymerase and the CytR repressor for the promoter region of the udp gene was shown. In the presence of the improved -10 region, the introduction of a substitution 15C-->T (that is the presence of the elongated Pribnow block) resulted in the CRP-independent transcription of the udp gene promoter. The binding site CRP2 was shown to be indispensable for the maximum promoter activation by the transcription-activating cAMP-CRP complex. Both positive (cAMP-CRP complex) and negative (CytR) regulation of the promoter was virtually fully abolished after the introduction of mutations leading to the creation of canonical sequences in -10 and -35 promoter regions.

[Determination of functional role of nucleotide composition in the transcription start region of the Escherichia coli udp gene]. / Opredelenie funktsional'noi znachimosti nukleotidnogo sostava v oblasti starta transkriptsii gena Eshcherichia coli.

The promoter of the Escherichia coli udp gene contains the poly-T (5'-TTTTT-3') motif in the transcription start region located at the distance of 3 nucleotides with respect to the Pribnow box. By means of site-directed mutagenesis, mutations in the +1, -1, and +3 positions of this region were isolated and their functional role in transcription initiation was determined. It was shown that in addition to the thymine nucleotide earlier identified at position 4 (with respect to the 5' end of the poly-T motif), the third thymine nucleotide may serve as an efficient transcription site. The functional significance of the presence of the poly-T motif in the transcription initiation region is discussed. Analysis of the isolated mutant promoters revealed the ability of the guanine nucleotide at the +3 position to stabilize the formation of a productive transcription complex and to serve as an additional transcription start.

[Functional interrelationship between elements of the Escherichia coli udp gene promotor responsible for binding regulatory proteins CytR, CRP, and RNA polymerase]. / Funktsional'noe perekryvanie élementov promotora gena udp Escherichia coli, otvetstvennykh za sviazyvanie reguliatornykh belkov CytR, CRP i RNK-polimerazy.

Site-directed mutagenesis was conducted in the regulatory region of the Escherichia coli udp gene at promoter sites responsible for binding regulatory proteins CRP and CytR as well as RNA polymerase (the core-promoter containing the--10 sequence). In mutants with an "improved"--10 region, a partial relief from the control of the cAMP-CRP transcription activation complex occurred, and the negative CytR repressor regulation was reduced. In contrast, mutant promoters with a weak Pribnow block or with a deletion that completely eliminates the core-promoter exhibited an increased ability to titrate the CytR protein in vivo. On the other hand, the affinity of CytR for DNA in mutants with an altered--10 region was the same as in the wild-type udp promoter. After introduction of mutations affecting binding sites for CRP (CRP1 and CRP2), the negative effect of the CytR protein on promoter transcription was fully abolished. The CRP1 binding site was shown to play the main role in the activation of the promoter by the cAMP-CRP complex, whereas the CRP2 site participates in the formation of the repressor complex. Mutations in the main and additional CytR binding sites were isolated and characterized. On the basis of these data, it is concluded that the modification of each structural element of the udp regulatory region (binding sites for CytR, CRP, or RNA polymerase) caused changes in the overall pattern of the promoter regulation.

[Protein engineering of uridine phosphorylase from Escherichia coli K-12. I. Cloning and expression of uridine phosphorylase genes from Klebsiella aerogenes and Salmonella typhimurium in E. coli]. / Belkovaia inzheneriia uridinofosforilazy iz Escherichia coli K-12. I. Klonirovanie i ékspressiia v E. coli genov uridinofosforilaz iz Klebsiella aerogenes i Salmonella typhimurium.

Genes of uridine phosphorylases (udp) from Klebsiella aerogenes and Salmonella typhimurium were cloned and expressed. Highly effective producer strains of the corresponding proteins were constructed. Enzymic properties of the UPases obtained were studied and compared with those from the Escherichia coli enzyme. Mutant forms of UPase from E. coli (D5E, D5N, D5A) were prepared by site-directed mutagenesis techniques. It was shown that the Asp5 residue plays an insignificant role in the formation of the active form of the protein.