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Termination Efficiency at Rho-Dependent Terminators Depends on Kinetic Coupling Between RNA Polymerase and Rho DING JUN JIN*T, RICHARD R

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Termination Efficiency at Rho-Dependent Terminators Depends on Kinetic Coupling Between RNA Polymerase and Rho DING JUN JIN*T, RICHARD R Proc. Nati. Acad. Sci. USA Vol. 89, pp. 1453-1457, February 1992 Biochemistry Termination efficiency at rho-dependent terminators depends on kinetic coupling between RNA polymerase and rho DING JUN JIN*t, RICHARD R. BURGESSt, JOHN P. RICHARDSON§, AND CAROL A. GROSS* *Department of Bacteriology and tMcArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI 53706; and §Department of Chemistry, Indiana University, Bloomington, IN 47405 Communicated by Peter H. von Hippel, November 4, 1991 ABSTRACT Rho-dependent terminators constitute one of nucleotides (19) shows increased termination at rho- two major classes of terminators in Escherichia coli. Termi- dependent terminators in vivo (20). The genetically identical nation at these sites requires the concerted action of RNA rpoB203 allele suppresses the termination defects of rho polymerase and rho protein. We present evidence that the mutants (21, 22). These experiments suggest that the effi- efficiency of termination at these sites is governed by kinetic ciency of rho to catalyze termination might be determined by coupling ofthe rate oftranscription ofRNA polymerase and the the rate of RNA polymerase elongation; that is, the two rate of action of rho protein. Termination experiments in vitro processes would be kinetically coupled. In this kinetic cou- indicate that termination efficiency at a rho-dependent termi- pling model, a slowly elongating RNA polymerase would nator is an inverse function of the rate of elongation of RNA terminate better than one that elongated more rapidly. polymerase, and each of the mutant phenotypes can be ac- To test whether termination efficiency is regulated by counted for by the altered rate ofelongation ofthe mutant RNA kinetic coupling, we took advantage oftwo RNA polymerase polymerase. Experiments in vivo show that fast-moving mutant mutants defective at rho-dependent termination: rpoB8 (in- RNA polymerases are termination deficient, while slow-moving creased termination) and rpoB3595 (decreased termination) mutant RNA polymerases are termination proficient and can (20). The altered termination characteristics ofthese mutants suppress the termination deficiency ofa slow-acting mutant rho can be explained by their altered rates of elongation. In protein. Because of the close coupling of rho action with RNA accord with the expectations of the kinetic coupling model, polymerase, small changes in the elongation rate of RNA the efficiency oftermination ofthe mutant RNA polymerases polymerase can have very large effects on termination effl- at rho-dependent terminators is inversely related to their ciency, providing the cell with a powerful way to modulate rates of elongation in vitro and in vivo. termination at rho-dependent terminators. MATERIALS AND METHODS Transcription termination is a regulated process that controls In Vitro Transcription. RNA polymerase (23) and rho (24) the activity of genes located downstream from terminators were purified as described. Transcription reactions were as and occurs at intrinsic or rho-dependent sites in Escherichia described (19). The standard assay conditions are lx NTPs coli (1-3). rho-dependent terminators share common features (0.2 mM each ATP, GTP, and CTP, and 0.02 mM UTP plus but lack a strong consensus sequence. A rho binding site is 5 ICi of [32P]UTP; 1 Ci = 37 GBq); other concentrations a region ofRNA rich in C residues and poor in G residues with indicate the relative change in each nucleotide compared to little secondary structure (4, 5), located =100 nucleotides the standard assay. Assays with rho were 15-min multiple- upstream of the multiple, closely spaced termination sites round transcriptions with 4 jig ofwild-type rho or 8 pug ofrhol characteristic of rho-dependent terminators (6-9). rho AT- per ml. The distribution of transcript molecules was quanti- Pase and helicase functions activated by binding to the RNA fied with the AMBIS Radioanalytic Imaging System (San transcript facilitate transcript release and dissociation of Diego). The data for each species were corrected for back- RNA polymerase (10, 11). rho-dependent termination sites ground and length differences and are expressed as percent- are typically RNA polymerase pause sites in the absence of age of total molecules (sum of molecules with 3' ends at rho (6-9). We have studied the effect of RNA polymerase on termination sites I, II, III, and readthrough). termination efficiency at rho-dependent terminators. 13-Galactosidase Assay. Cells growing exponentially in M9 There is no biochemical evidence that RNA polymerase glycerol (0.2%) medium supplemented with all amino acids and rho interact structurally. rho does not bind to core RNA and bases (25) were induced for lacZ expression with 1 mM polymerase (12); if these proteins interact, a special confor- isopropyl 83-D-thiogalactoside at A450 = 0.3. Duplicate sam- mation in the ternary complex is required for this interaction ples (0.5 ml each) were withdrawn every 15 sec into tubes to occur. However, some RNA polymerase mutations are containing 0.5 ml of ice-cold chloramphenicol (0.1 mg/ml) to allele-specific suppressors of the termination defects of stop protein elongation. After sampling, the tubes were strains with mutant rho alleles (13-15), suggesting that the incubated for 15 min at 370C to allow the synthesized 8-ga- two proteins at least interact functionally. lactosidase monomers time to assemble. A3-Galactosidase A kinetic mechanism for this functional interaction is was measured as described (25). suggested by several observations. RNA polymerase with decreased pausing because of the ri S01 mutation (16) or RESULTS addition of the AQ antitermination protein (17) has an in- Efficiency of Termination with rho in Vitro Can Be Altered creased rate of elongation and a decreased termination effi- by Changing the Rate of Elongation of RNA Polymerase. A ciency at rho-dependent terminators in vitro. The RpoB8 prediction of the kinetic coupling model is that mutant and RNA polymerase with increased pausing (18) and a reduced wild-type RNA polymerase will have comparable extents of elongation rate because it is defective in binding purine termination at comparable rates of transcription elongation. The publication costs of this article were defrayed in part by page charge tPresent address: Building 37, Room 1E16, Laboratory of Molecular payment. This article must therefore be hereby marked "advertisement" Biology, National Cancer Institute, National Institutes of Health, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Bethesda, MD 20892. 1453 Downloaded by guest on September 30, 2021 1454 Biochemistry: Jin et al. Proc. Natl. Acad. Sci. USA 89 (1992) To determine whether this is true , we examined their ability simply a consequence of their altered elongation rates. That to terminate in vitro at the best-sstudied rho-dependent ter- RpoB8 has somewhat lower readthrough than wild type is minator, AtR1, initially characteri2zed by Rosenberg et al. (26) consistent with our previous findings that, under these con- and Court et al. (27). With rho, trranscription initiates at the ditions, the elongation rate ofthe mutant approaches but does APR promoter and terminates at ane of three closely spaced not quite reach that of the wild type (19). sites (I, II, III). In the absence of rho, RNA polymerase These experiments also demonstrate the profound effect of pauses at these sites (6-9). the rate of elongation of RNA polymerase on the efficiency To compare termination efficic ncies at comparable elon- and pattern of rho-dependent termination. Comparing termi- gation rates of RNA polymerases, it is necessary to establish nation at 0.25x and 4x nucleotides for the wild-type enzyme how the elongation rate of each RINA polymerase varies with (Fig. 2, lanes 1 and 5) indicates that there is a 5-fold increase nucleotide concentration. For Rj)oB8, a 4-fold higher con- in the extent of readthrough of the AtR1 terminator. In centration of nucleotides gives an elongation rate approach- addition, the pattern of termination changes. When RNA ing that of the wild type (19). We used a single-round polymerase moves slowly, most termination is at termination transcription assay from APR tc Z establish the nucleotide site I, whereas when RNA polymerase moves rapidly, ter- dependence for RpoB359S. RpoB13595 elongates faster than mination at the downstream termination sites becomes more the wild type (Fig. 1, compare laties 1-3 to lanes 10-12) but important. Morgan et al. (8, 9) also found that pausing and exhibits a comparable rate of elon~gation when it has a 2-fold termination are enhanced at site I of AtR1 when nucleotide lower concentration of nucleotidles (compare lanes 1-3 to concentration is reduced. Both the inverse relationship be- lanes 7-9 and lanes 4-6 to lanes 10-12). tween rate of elongation of an RNA polymerase and effi- The pattern of rho-mediated ter.7minationSforforwild-typethe and wouldciency ofbeterminationexpected if,andasthepredictedchange inbypatternthe kineticoftermination both mutant RNA polymerases as coupling tion rate is presented in Fig. 2A. T'hese data, tabulated in FigFi. model, the efficiency of rho to catalyze termination were 2B, indicate that terminations by determined by the rate of RNA polymerase elongation. iheseudatantabuldmutant and wild-typenldtypRNA Suppression of a Mutant rho Allele by rpoB8 Can Be polymerases at comparable rates of elongation are virtually Explaied by Its Slow Elongation Rate. A second phenotype identical.
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