Affinity Labelling of the Active Center of DNA-dependent RNA Polymerases within the Archaebacterial Kingdom

Abstract

SummaryThe super-selective affinity labelling procedure for the active center of RNA Polymerase from Escherichia coli (Grachev et al., 1987a) was successfully applied to the enzyme from archaebacteria. Using adenosine-5'-trimetaphosphate or the p-hydroxybenzaldehyde ester of ATP, the second largest subunit B' of the RNA polymerase from methanogenic/halophilic and sulphate reducing archaebacteria, and the largest subunit B of the non-methanogenic thermophilic archaebacterium Sulfolohus sp. strain B 12 are labelled specifically. The labelling reaction is strictly template-dependent and blocked by the transcription inhibitor heparin. We present evidence that adenosine-5'-trimetaphosphate is attached close to the catalytic center of the RNA polymerase via a phosphoamide bond to lys or his residues.The specific affinity labelling of subunit B' from methanogens/halophiles together with the earlier observed immunological cross-reactivity (Gropp et al., 1986) indicates that this subunit as well as subunit B" contains amino acid sequences which are homologous to sequences in subunit B of the enzyme from non-methanogenic thermophilic archaebacteria and in subunit ß from E. coli RNA polymerase.

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

“…Reagents I, III, VI and VIII are the most efficient, whereas all the rest produced only very weak labeling. It is noteworthy that the same reagents are among the most efficient in affinity labeling of RNA polymerase from E. coli and wheat germ [1,51. In all of these cases, the B-subunit, which was also identified earlier [7] as the major target of reagent VI, became labeled. This subunit is immunologically [14] and structurally homologous to the second largest subunit of eukaryotic and prokaryotic RNA polymerases.…”

“…Hartmann, Institut fiir Biochemie der Ludwig-Maximilians-Universit~it, Karlstr. 23, D-8000 Miinchen 2, FRG and archaebacterial RNA polymerases [5][6][7][8][9][10] suggested that the binding site of the initiating substrate resides on the g?-subunit of prokaryotic enzymes, on the second largest subunit of eukaryotic forms and on the B-or B'-subunit of the archaebacterial types. Comparison of the amino acid sequences close to the labeled sites may shed some light on the conservation of these regions during evolution.…”