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Volume 271, Number 24, Issue of June 14, 1996 pp. 14572-14583
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Amino Acid Substitutions in the Two Largest Subunits of Escherichia coli RNA Polymerase That Suppress a Defective Rho Termination Factor Affect Different Parts of the Transcription Complex

(Received for publication, December 18, 1995, and in revised form, March 27, 1996)

Laura M. Heisler , Guohua Feng ^¶ , Ding Jun Jin ^'' , Carol A. Gross and Robert Landick ^¶

From the  Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706, the ^¶ Department of Biology, Washington University, St. Louis, Missouri 63130 and the ^'' Laboratory of Molecular Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892

Among the earliest rpoBC mutations identified are three suppressors of the conditional lethal rho allele, rho201. These three mutations are of particular interest because, unlike rpoB8, they do not increase termination at all -dependent and -independent terminators. rpoB211 and rpoB212 both change Asn-1072 to His in conserved region H of rpoB (N1072H), whereas rpoC214 changes Arg-352 to Cys in conserved region C of rpoC (R352C). Both substitutions significantly reduce the overall rate of transcript elongation in vitro relative to wild-type RNA polymerase; however, they probably slow elongation for different reasons. The nucleotide triphosphate concentrations required at the T7 A1 promoter for both abortive trinucleotide synthesis and for promoter escape are much greater for N1072H. In contrast, R352C and two adjacent substitutions (G351S and S350F), but not N1072H, formed open complexes of greatly reduced stability. The sequence in this region of modestly resembles a region of Escherichia coli DNA polymerase I that contacts the phosphate backbone of DNA in co-crystals. Core determinants affecting open complex formation do not reside exclusively in , however, since the Rif^r mutation rpoB2 in  also dramatically destabilized open complexes. We suggest that the principal defects of the two Rho-suppressing substitutions may differ, perhaps reflecting a greater role of  region H in nucleoside triphosphate-binding and nucleotide addition and of region C in contacts to the DNA strands that could be important for translocation. Although both probably suppress rho201 by slowing RNA chain elongation, these differences may lead to terminator specificity that depends on the rate-limiting step at different sites.

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