Biomedical subjects
Publications and source records attributed to V Sagitov.
The rate of transcription elongation in Escherichia coli was reduced when cells were depleted of NusG. In a purified in vitro system, NusG accelerated the transcription elongation rate. The stimulation of the rate of transcription elongation by NusG appears to result from the suppression of specific transcription pause sites.
The rrnB P1 promoter of Escherichia coli (starting sequence C-4-A-3-C-2-C-1-A+1-C+2-U+3-G+4) forms a binary complex with RNA polymerase that is highly unstable and requires the presence of transcription substrates ATP and CTP for stabilizing the enzyme-DNA association (Gourse, R. L. (1988) Nucleic Acids Res. 16, 9789-9809). We show that in the absence of UTP and GTP the stabilization is accomplished by short RNA oligomers synthesized in an unusual "-3-->" mode whereby the primer initiated at the +1 site presumably slips back by three nucleotides into the -3 site and is then extended yielding stable ternary complexes. By contrast, short oligomers initiated in the conventional "+1-->" mode without slippage do not exert the stabilization effect and are readily aborted from the promoter complex. The stable -3-->ternary complexes carry sigma factor but otherwise resemble elongation complexes in their high salt stability and in the fact that they are formed with a mutant RNA polymerase deficient in promoter binding. A model is proposed explaining the stability of the -3-->ternary complexes by RNA slipping into a putative "tight RNA binding site" in RNA polymerase which is normally occupied by RNA during elongation.
Two transcription elongation factors (GreA and GreB) related in primary sequence were isolated from E. coli. Each factor induced cleavage of the nascent transcript in artificially halted elongation complexes followed by the loss of the 3' proximal fragment and resumption of elongation from the new 3' terminus. GreA induced cleavages 2 or 3 nt behind the terminus while GreB released longer oligonucleotides up to 9 nt in length. The pattern of cleavages characteristically changed as the transcription complex advanced, supporting the "inchworm" model of RNA polymerase propagation. In addition to attacking artificially halted complexes, both factors antagonized the action of natural elongation-arresting sites that occasionally trap the advancing complex. GreB rescued the arrested complexes via the transcript cleavage and restart pathway while GreA acted by an unknown mechanism, preventing the arrest only if added before the polymerase reached the arresting site.
The segment Asp1064-Lys1073 in the beta subunit of Escherichia coli RNA polymerase is evolutionarily conserved and is located near the "5' face" of the nucleotide binding pocket as was shown by affinity labeling with priming substrates (Grachev, M. A., Lukhtamov, E. A., Mustaev, A. A., Zaychikov, E. F., Abdukayumov, M. N., Rabinov, I. V., Richter, V. I., Skoblov, Y. S., and Chistyakov, P. G. (1989) Eur. J. Biochem. 180, 577-585). We engineered single Xaa-->Ala or Ala-->Ser substitutions of eight evolutionarily conserved amino acids in this segment as well as a multiple alanine (KRNK) substitution of four of these residues. The KRNK mutation as well as four of the single substitutions were dominant lethal, two of the single mutations were recessive lethal, and two were viable. RNA polymerase bearing the dominant mutations was prepared for biochemical study by in vitro reconstitution from subunits. All of the mutant enzymes formed stable, specific promoter complexes, capable of initiating RNA synthesis. However, the KRNK polymerase was totally blocked in initiation-to-elongation transition, whereas the four point mutants displayed allele-specific changes in promoter clearance rate. Each of the four mutations changed, in a specific way, both the pattern of short oligomers generated in abortive initiation and the pattern of RNA polymerase pausing during elongation. Thus, the mutations appear to distort but not destroy the active center and to alter, in allele-specific manner, the coupling between the catalytic reaction and RNA polymerase propagation along the template.
Deletion of 10 amino acids from a conserved motif in the beta subunit of Escherichia coli RNA polymerase (RNAP) leads to an interrupted transcription cycle and lethal phenotype. RNAP carrying the mutant subunit retains catalytic function and specificity of promoter recognition but is unable to efficiently hold onto DNA in the binary complex, resulting in a diminished initiation frequency. However, inefficient initiation by the mutant enzyme leads to processive and stable ternary elongating complex. Thus, the mutation dissects the traits of promoter selectivity, binary complex stability, and ternary complex processivity reflecting compartmentalization of function within the RNAP molecule.