ELECTRON-MICROSCOPY MAPPING OF ESCHERICHIA-COLI RNA POLYMERASE-BINDING SITES ON PLASMIDS FROM THERMOPHILIC BACTERIA
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1984
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Abstract
The binding sites of E. coli RNA polymerase to plasmid DNA from extremely thermophilic bacteria were mapped by EM. Templates used in these studies included plasmids pTF62 (from Thermus flavus AT62) and pTT8 (from T. thermophilus HB8) and hybrid molecules constructed by ligation of these plasmids to pBR322. Although the affinity of the enzyme for heterologous DNA was .apprx. 1/3 of that for pBR322, it was possible to localize preferred binding sites on pTF62 and pTT8. Six binding sites were identified in pTT8, mapping close to 7, 28, 47, 61, 65 and 81 map units (1 unit = 1% of the length of the DNA). Seven such regions located at 3, 27, 48, 60, 67, 81 and 86 map units were found in pTF62. RNA polymerase binding sites found in pBR322 coincided with promoters identified previously by EM analysis of transcriptional complexes prepared in vitro. Evidently, E. coli RNA polymerase binds preferentially to specific sequences in plasmids from thermophilic bacteria, suggesting possible promoter locations in these plasmids.