Endonucleolytic Function of MutLα in Human Mismatch Repair

The path of the nucleic acids through a transcription elongation complex was tracked by mapping cross-links between bacterial RNA polymerase (RNAP) and transcript RNA or template DNA onto the x-ray crystal structure. In the resulting model, the downstream duplex DNA is nestled in a trough formed by the β′ subunit and enclosed on top by the β subunit. In the RNAP channel, the RNA/DNA hybrid extends from the enzyme active site, along a region of the β subunit harboring rifampicin resistance mutations, to the β′ subunit “rudder.” The single-stranded RNA is then extruded through another channel formed by the β-subunit flap domain. The model provides insight into the functional properties of the transcription complex.

https://science.sciencemag.org/content/sci/289/5479/619.full.pdf

A Structural Model of Transcription Elongation

https://www.cell.com/molecular-cell/pdf/S1097-2765(07)00374-7.pdf

A Central Role of the RNA Polymerase Trigger Loop in Active-Site Rearrangement during Transcriptional Pausing

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 β subunit.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1988.tb14384.x

Genes coding for RNA polymerase β subunit in bacteria

The elongation of RNA chains during transcription occurs in a ternary complex containing RNA polymerase (RNAP), DNA template, and nascent RNA. It is shown here that elongating RNAP from Escherichia coli can switch DNA templates by means of end-to-end transposition without loss of the transcript. After the switch, transcription continues on the new template. With the use of defined short DNA fragments as switching templates, RNAP-DNA interactions were dissected into two spatially distinct components, each contributing to the stability of the elongating complex. The front (F) interaction occurs ahead of the growing end of RNA. This interaction is non-ionic and requires 7 to 9 base pairs of intact DNA duplex. The rear (R) interaction is ionic and requires approximately six nucleotides of the template DNA strand behind the active site and one nucleotide ahead of it. The nontemplate strand is not involved. With the use of protein-DNA crosslinking, the F interaction was mapped to the conserved zinc finger motif in the NH2-terminus of the β′ subunit and the R interaction, to the COOH-terminal catalytic domain of the β subunit. Mutational disruption of the zinc finger selectively destroyed the F interaction and produced a salt-sensitive ternary complex with diminished processivity. A model of the ternary complex is proposed here that suggests that trilateral contacts in the active center maintain the nonprocessive complex, whereas a front-end domain including the zinc finger ensures processivity.

https://science.sciencemag.org/content/sci/273/5272/211.full.pdf

Transcription processivity: protein-DNA interactions holding together the elongation complex.

A method is proposed for localization of the sites of affinity labelling of the β subunit of Escherichia coli RNA polymerase. The principle of this method is similar to that of the methods of rapid sequencing of nucleic acids.



The polypeptide bearing a radioactive affinity label at one of the amino acid residues is subjected to short-term treatment with cyanogen bromide. The conditions of this reaction are selected in such a way that less than one cleavage occurs on average per polypeptide chain. Two series of radioactive peptides are formed, one involving all the possible N-terminal peptides and the other the C-terminal peptides. The distribution of the lengths of these peptides is studied by means of gel electrophoresis and compared with the theoretical ones based on the known amino acid sequence of the β subunit. Obviously, the affinity label resides between the C-terminus of the shortest N-terminal radioactive peptide and the N-terminus of the shortest C-terminal radioactive peptide. In order to increase reliability and resolution of the method, partial trypsinolysis may be employed.



The evidence obtained suggests that lysine residues over the regions 1036–1066, 1234–1242, and histidine-1237 are situated in the nearest neighbourhood to, or directly involved in the formation of the active center of initiating substrate binding of the β subunit of E. coli RNA polymerase.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1989.tb14684.x

Studies on the functional topography of Escherichia coli RNA polymerase. Highly selective affinity labelling by analogues of initiating substrates.

A method is proposed for localization of the sites of affinity labelling of the beta subunit of Escherichia coli RNA polymerase. The principle of this method is similar to that of the methods of rapid sequencing of nucleic acids. The polypeptide bearing a radioactive affinity label at one of the amino acid residues is subjected to short-term treatment with cyanogen bromide. The conditions of this reaction are selected in such a way that less than one cleavage occurs on average per polypeptide chain. Two series of radioactive peptides are formed, one involving all the possible N-terminal peptides and the other the C-terminal peptides. The distribution of the lengths of these peptides is studied by means of gel electrophoresis and compared with the theoretical ones based on the known amino acid sequence of the beta subunit. Obviously, the affinity label resides between the C-terminus of the shortest N-terminal radioactive peptide and the N-terminus of the shortest C-terminal radioactive peptide. In order to increase reliability and resolution of the method, partial trypsinolysis may be employed. The evidence obtained suggests that lysine residues over the regions 1036-1066, 1234-1242, and histidine-1237 are situated in the nearest neighbourhood to, or directly involved in the formation of the active center of initiating substrate binding of the beta subunit of E. coli RNA polymerase.

Arkady A. Mustaev

Papers

Studies on the functional topography of Escherichia coli RNA polymerase. Highly selective affinity labelling by analogues of initiating substrates.

Studies of the functional topography of Escherichia coli RNA polymerase. A method for localization of the sites of affinity labelling.