The Primary Structure of Escherichia coli RNA Polymerase

Abstract

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...

Translational Barrier in Central Region of Encephalomyocarditis Virus Genome

“…Firstly, highly purified preparations of EF-X exhibited two functional activities of eEF-2: the ability to promote the synthesis of polyphenylalanine by poly(U)-programmed washed ribosomes in the presence of eEF-1 and aminoacylated tRNA, and to undergo ADP-ribosylation in the presence of NAD and diphtheria toxin. Moreover, these two activities were found to co-purify, upon respectively [33,34] Sephacryl S-300 and phosphocellulose chromatography, with both the EF-X activity and p105 (compare Table 1 with Fig. 3 and 4).…”

Cyclic GMP phosphodiesterase from bovine retina Amino acid sequence of the α‐subunit and nucleotide sequence of the corresponding cDNA

“…The nucleotide sequence of DNA fragments was determined according to Maxam and Gilbert [7] and Sanger et al [8]. The strategy of determining the nucleotide sequence was analogous to that used in [9].…”

Localisation of the binding site for the initiating substrate on the RNA polymerase from Sulfolobus acidocaldarius

“…Comparison of amino acid sequence near the labeled region of RNA polymerase subunit B from S. acidocaldarius and subunit from E. coli [15] with similar sequences present in the second largest subunit of RNA polymerase B (II) from yeast [l 1] and Drosophila [12] and in subunit B' of the enzyme from H. halobium [16] and M. thermoautotrophicum [17]. Number indicates position of the first amino acid of the sequence.…”

“…subunit of archaebacterial RNA polymerase from S. acidocaldarius which is highly homologous to a region in the fl-subunit of E. coli RNA polymerase [15] which becomes labeled by the same reagent ( fig.5). Furthermore, this region is also homologous to a sequence found in subunit B' of RNA polymerase from the archaebacteria Halobacterium halobium [16] and Methanobacterium thermoautotrophicum [17] and in the second largest subunit of RNA polymerase B (II) from yeast [11] and Drosophila [12].…”