ROLE OF THE SIGMA(70)-SUBUNIT OF ESCHERICHIA-COLI RNA-POLYMERASE IN TRANSCRIPTION ACTIVATION

被引:98
作者
KUMAR, A
GRIMES, B
FUJITA, N
MAKINO, K
MALLOCH, RA
HAYWARD, RS
ISHIHAMA, A
机构
[1] NATL INST GENET, DEPT MOLEC GENET, MISHIMA, SHIZUOKA 411, JAPAN
[2] UNIV EDINBURGH, INST CELL & MOLEC BIOL, EDINBURGH EH9 3JR, MIDLOTHIAN, SCOTLAND
[3] OSAKA UNIV, MICROBIAL DIS RES INST, SUITA, OSAKA 565, JAPAN
基金
英国惠康基金;
关键词
RNA POLYMERASE; SIGMA SUBUNIT; TRANSCRIPTION FACTOR; PROTEIN PROTEIN CONTACT; TRANSCRIPTION REGULATION;
D O I
10.1006/jmbi.1994.1001
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The role of the σ70 subunit of Escherichia coli RNA polymerase in transcription activation by positive transcription factors was investigated. For this purpose, we constructed a nested set of E. coli rpoD deletion mutants generating carboxy-terminally truncated σ70 subunits of RNA polymerase in a high-expression plasmid. The purified mutant σ70 subunits were reconstituted into holoenzymes and examined in vitro for their promoter selectivity. As expected, since the -35 recognition helix of σ70 was deleted in all cases, the mutant enzymes were unable to initiate at factor-independent promoters, except for the special case of perfect “extended minus 10” promoters, at which the need for -35 sequence recognition by RNA polymerase is replaced by recognition of additional base-pairs in the -10 region. However, two factor-dependent promoters, PhoB-dependent PpstS and cAMP receptor protein (CRP)-dependent P1gal, could be activated for transcription by different subsets of the mutant holoenzymes, although these promoters do not contain the perfect extended -10 sequences. These results establish that -35 DNA recognition by σ70 is not essential for these cases. Presumably it is replaced by protein-protein contacts between RNA polymerase and the activator, which in both cases is bound to the DNA in a position overlapping the -35 region. Further, the detailed results support the view that the contact and/or activation sites for these two factors may lie on the σ70 subunit, within a “contact site II”, which extends at least from conserved region 3.2 to the upstream end of region 4.2. Moreover, as in the case of contact site I on the α subunit, it appears that contact site II contains various different subsites for interaction with specific class II activators, and that PhoB and CRP require distinct subsites. © 1994 Academic Press Limited.
引用
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页码:405 / 413
页数:9
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