The nucleotide sequence of the rpoB gene of Salmonella typhimurium has been determined in this work. It was compared with known sequences of the gene from other sources and the conservative regions were detected. This allowed some interesting conclusions to be made about the distribution of the functional domains in bacterial RNA polymerase and about the three-dimensional structure of its p subunit.DNA-dependent synthesis of all types of RNA in bacteria is catalyzed by RNA polymerase, an oligomeric enzyme with the subunit structure .$j?'o (for review see [l]). To study the structure and the mechanism of RNA polymerase action, amino acid sequences of all subunits of the E. coli enzyme were determined [2-51, the subunit in contact with the DNA template, RNA product and substrates were identified [6] and several mutations leading to RNA polymerase resistance to the antibiotics rifampicin and streptolydigin were localized [7 -101. These investigations indicated an important role for the RNA polymerase p subunit at all stages of RNA synthesis. This paper presents the results of cloning and sequencing of the rpoB gene coding for the subunit of Salmonella typhimurium RNA polymerase.The primary structure of the Salmonella typhimurium rpoB gene coding for RNA polymerase p subunit was determined (4.2 kb in toto). On comparison with homologous Escherichia coli and tobacco chloroplasts gene sequences, nine conservative and six variable regions of the [ j subunit structure were identified. Analysis of available genetic data allowed some preliminary conclusions to be made concerning the location of the putative functional domains in the structure of the p subunit of bacterial RNA polymerase and to devise a hypothesis about the spatial proximity of the central part of the p subunit to its N-and C-terminal domains.
The combined structural study of proteins and of their corresponding genes utilizing the methods of both protein and nucleotide chemistry greatly accelerates and considerably simplifies both the nucleotide and protein structure determination and, in particular, enhances the reliability of the analysis. This approach has been successfully applied in the primary structure determination of the fl and j' subunits of Escherichia coli DNAdependent RNA potynierase and of their structural genes, yielding a continuous nucleotide sequence (4714 base pairs) that embraces the entire rpoB gene, the initial part of the rpoC gene and the intercistronic region, together with the total amino acid sequence of the fl subunit, comprising 1342 residues, and the N-terminal sequence of the jl' subunit (1 76 residues).Elucidation of the transcription mechanism requires detailed knowledge of the active-center organization of RNA polymerase at the various stages of the RNA synthesis. This, in turn, can be obtained only after determining the primary and spatial structure of the enzyme.Earlier we had established the amino acid sequence of the x subunit of Escherichiu coli DNA-dependent RNA polymerase by resorting solely to the ordinary methods of protein chemistry [5]. In the case of the fl and p' subunits with their much higher molecular weights (= 155000 and z 165000, respectively) [6], such an approach could no longer suffice, in view of the difficulties in isolating and purifying the resulting fragments and in reconstituting the amino acid sequence via overlapping peptides [7].The progress in DNA sequencing methods and the possibility of using the genetic code to obtain information on the primary protein structure from the nucleotide sequences is an attractive way to circumvent such difficulties; although here, too, there are many pitfalls, requiring considerable caution to avoid possible sources of error.In the first place the mRNA can undergo processing, leading to erroneous deduction of the protein structure. Secondly, the protein itself can be processed. Thirdly, it is often difficult to recognize in the overall DNA structure the beginning of a structural gene. The criterion for this purpose is the presence of an initiating codon together with the adjacent sequences complementary to the 3' end of 16-S RNA [8,9]. Frequently more than one such combination can be found for one and the same protein. insertion) in the DNA sequence determination could lead to a completely erroneous amino acid sequence of the protein. Thus, primary structure determination of DNA cannot serve as a substitute for the direct sequencing of the protein.In view of this, we decided to utilize the methods of both protein and nucleotide chemistries, performing the parallel sequencing of the structural genes rpoB (jl subunit) and rpoC (jl' subunit) and of the corresponding proteins. Knowledge of the nucleotide sequence of the pertinent DNA segments would permit aligning of the peptide fragments from the protein analysis into an uninterrupted polypeptide chain. Such...
Five recombinant plasmids, pBK2646, pBK611, pRC3, pRC4 and pRC5, carrying rpoB rifampicin-resistant RNA-polymerase genes were obtained. The sequence analysis of these plasmids revealed certain structural changes in the rpoB gene which specify corresponding alterations in the beta-subunit of RNA polymerase. Some functional properties of the corresponding mutant strains and their RNA polymerases have been investigated.
(Na+ + K+)‐ATPase α‐Subunit β‐Subunit cDNA nucleotide sequence Primary structure Glycopeptide Transmembrane arrangement
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