Escherichia coli promoter -10 and -35 region homologies correlate with binding and isomerization kinetics.
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ABSTRACT:
SUBMITTER: Studnicka GM
PROVIDER: S-EPMC1149221 | biostudies-other | 1988 Jun
REPOSITORIES: biostudies-other
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Project description:The sigmas subunit of Escherichia coli RNA polymerase holoenzyme (EsigmaS) is a key factor of gene expression upon entry into stationary phase and in stressful conditions. The selectivity of promoter recognition by EsigmaS and the housekeeping Esigma70 is as yet not clearly understood. We used a genetic approach to investigate the interaction of sigmaS with its target promoters. Starting with down-promoter variants of a sigmaS promoter target, osmEp, altered in the -10 or -35 elements, we isolated mutant forms of sigmaS suppressing the promoter defects. The activity of these suppressors on variants of osmEp and ficp, another target of sigmaS, indicated that sigmaS is able to interact with the same key features within a promoter sequence as sigma70. Indeed, (i) sigmaS can recognize the -35 element of some but not all its target promoters, through interactions with its 4.2 region; and (ii) amino acids within the 2.4 region participate in the recognition of the -10 element. More specifically, residues Q152 and E155 contribute to the strong preference of sigmaS for a C in position -13 and residue R299 can interact with the -31 nucleotide in the -35 element of the target promoters.
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Project description:Classical elements of ?(70) bacterial promoters include the -35 element ((-35)TTGACA(-30)), the -10 element ((-12)TATAAT(-7)), and the extended -10 element ((-15)TG(-14)). Although the -35 element, the extended -10 element, and the upstream-most base in the -10 element ((-12)T) interact with ?(70) in double-stranded DNA (dsDNA) form, the downstream bases in the -10 motif ((-11)ATAAT(-7)) are responsible for ?(70)-single-stranded DNA (ssDNA) interactions. In order to directly reflect this correspondence, an extension of the extended -10 element to a so-called -15 element ((-15)TGnT(-12)) has been recently proposed. I investigated here the sequence specificity of the proposed -15 element and its relationship to other promoter elements. I found a previously undetected significant conservation of (-13)G and a high degeneracy at (-15)T. I therefore defined the -15 element as a degenerate motif, which, together with the conserved stretch of sequence between -15 and -12, allows treating this element analogously to -35 and -10 elements. Furthermore, the strength of the -15 element inversely correlates with the strengths of the -35 element and -10 element, whereas no such complementation between other promoter elements was found. Despite the direct involvement of -15 element in ?(70)-dsDNA interactions, I found a significantly stronger tendency of this element to complement weak -10 elements that are involved in ?(70)-ssDNA interactions. This finding is in contrast to the established view, according to which the -15 element provides a sufficient number of ?(70)-dsDNA interactions, and suggests that the main parameter determining a functional promoter is the overall promoter strength.
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Project description:In bacteria, promoter identification by RNA polymerase is mediated by a dissociable ? factor. The housekeeping ?(70) factor of Escherichia coli recognizes two well characterized DNA sequence elements, known as the '-10' and '-35' hexamers. These elements are separated by 'spacer' DNA, the sequence of which is generally considered unimportant. Here, we use a combination of bioinformatics, genetics and biochemistry to show that ?(70) can sense the sequence and conformation of the promoter spacer region. Our data illustrate how alterations in spacer region sequence can increase promoter activity. This stimulatory effect requires ?(70) side chain R451, which is located in close proximity to the non-template strand at promoter position -18. Conversely, R451 is not required to mediate transcriptional stimulation by improvement of the -10 element. Mutation of ?(70) residue R451, which is highly conserved, results in reduced growth rate, consistent with a central role in promoter recognition.
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