Immunoblotting of size-separated whole cell proteins permitted the study of protein-protein interaction. Briefly, proteins obtained from cleared cell lysates of Escherichiu coli were separated by glycerol gradient centrifugation and analysed by blotting against a set of specific antibodies. We have applied this procedure to the assembly of 11 N-terminal amber fragments of the fl subunit of E. coli RNA polymerase ranging in size between 97% and 23% the length of the intact fl polypeptide (1342 amino acids). In this way, we have been able to define regions on the fl polypeptide involved in the assembly of RNA polymerase.RNA polymerase of Escherichiu coli is a multimeric enzyme consisting of at least four different polypeptides, a, fl, p' and one sigma subunit (reviewed in [l, 21). Two main forms of the enzyme are present, core (a2flP') and holoenzyme ( U~~P ' G ) ; the (i factor in holoenzyme is required for specific transcription at promoter sites. This oligomeric complex is assembled in a defined pathway: a2 + uzfl + uzp/?' + a 2 P P '~, resulting in the production of intermediates during the biogenesis of the active holoenzyme (reviewed in [3,4]). The assembly of these subunits is an absolute requirement for the expression of intrinsic activities associated with RNA polymerase. In addition, the specificity of RNA polymerase, particularly its promoter selectivity, is controlled by G subunit exchange or the association of particular transcription factors. An understanding of protein-protein interaction involved in RNA polymerase assembly is therefore of paramount importance for interpreting structure-function characteristics of this multimeric enzyme.Many techniques exist for the study of protein-protein interaction but they suffer from the complexity and/or the invasiveness of the procedure. We have developed a rapid and sensitive technique that combines two existing procedures, glycerol gradient centrifugation under non-denaturing conditions and immunoblotting. Briefly, the protein complex(es) under study (in this case we are dealing with proteins isolated from E. coli after gentle lysis) are separated by centrifugation in a glycerol gradient [5], and each fraction is analysed by SDS/polyacrylamide gel electrophoresis followed by immunoblotting [6] against antisera for one of the components of the protein complex. The same nitrocellulose filter can be used for immunostaining consecutively against a series of different antibodies. By this procedure, it is possible to separate protein complexes according to size and to determine the fate of each protein component. As an initial attempt to determine the sites (or domains) on each RNA polymerase subunit involved in subunit-subunit interaction, we have employed this procedure to analyse the in vivo assembly of amber fragments of E. coli RNA polymerase. We have isolated previously 95 independent, spontaneously occurring mutants carrying amber mutations affecting expression of the fl structural gene, rpoB, and have identified the N-terminal amber fragments in 50 of th...

A 1.5 kb EcoRI-BamHI restriction fragment from Mycobacterium tuberculosis was found to hybridize specifically with genomic DNA from M. tuberculosis-complex organisms. Primers were designed from the terminal sequences of this fragment and used to amplify uniquely M. tuberculosis-group DNA in a polymerase chain reaction. It is suggested that a combination of these primers and probe will prove a useful tool for the early diagnosis of tuberculous infections.

No abstract

No abstract