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

The 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 Sulfolobus 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 imrnunological 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 β from E. coli RNA polymerase.