A. Kumar et al., A HYBRID SIGMA-SUBUNIT DIRECTS RNA-POLYMERASE TO A HYBRID PROMOTER INESCHERICHIA-COLI, Journal of Molecular Biology, 246(5), 1995, pp. 563-571
Most of the sigma (transcriptional initiation specificity) subunits of
RNA polymerase, from a wide range of eubacteria, show strong elements
of amino acid sequence similarity. There is evidence that two of the
''conserved'' regions, 2.4 and 4.2, are involved in recognition of the
consensus DNA sequences centred near -10 and -35, respectively, which
define promoter sites for the initiation of transcription. Since all
the alternative sigma subunits of the above type function by binding t
o a common core polymerase enzyme in a given bacterium, it can be pred
icted that a hybrid sigma might be functional, and if so should permit
RNA polymerase to initiate only at a correspondingly hybrid promoter.
To test these predictions, a hybrid gene encoding the amino-proximal
529 amino acids of the major Escherichia coli sigma protein, sigma(70)
(including region 2.4) followed by the last 82 amino acids of the hea
t-shock sigma protein, sigma(32) (including region 4.2) was constructe
d and fused to Plac on a plasmid. Major-consensus, heat-shock and hybr
id promoters were fused to a chloramphenicol acetyl transferase (CAT)
reporter gene on a compatible plasmid. CAT assays showed that, as pred
icted, a promoter with a ''heat-shock'' -35 consensus and a ''major''
-10 consensus sequence (P-HM) required Pine-dependent production of th
e hybrid sigma (sigma(70-32)) for activity in vivo. P-HM then became a
strong promoter. The hybrid sigma gene has potential advantages over
its parents for structure-function studies.