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. At the slowest rate of el *onoRot2 of the rpoB8 mutation is its altered interaction with mutant and RpoB8 at 1x nucleotides), bol theea rho alleles. As originally determined by Guarente and Beck- est termination at site I and compahablenzm showntsoof with is readthrough transcript. At a higheirraterabefofealontsongation (RPOBth+ (22)] (21),suppressesrpoB203the[whichterminationgeneticallydefect ofidenticalthe rho201to rpoB8muta- at 0.Sx, RpoB8 at 2x, and RpoB 355at0tion. To further define the mechanism of suppression, we all three enzymes terminate predominantly at site I but show examined the interaction of rpoB8 with rho) (suAl) originally increased readthrough. At a still I high~er ra~te of elongation isolated as a polarity suppressor of nonsense mutations (28). (RpoB+ at lx, RpoB8 at 4x, and RpoB3595 at 0.5 x nucleo- rho) encodes a mutant protein that binds to nascent RNA tides), all three enzymes use tihe three termination sites with normal kinetics but is slow in RNA release in vitro (29). relatively equally and show still grneater readthrough. The fact If this mutant rho protein also functions more slowly than that wild type and mutant show sicnilar termination profiles at wild-type rho in vivo, the kinetic coupling model led us to similar elongation rates strongly su lpports the proposition that predict that the slowly elongating RpoB8 RNA polymerase the altered termination phenotypes ofthe mutant enzymes are might suppress the termination defect of rhol by resynchro-

A. RpoB B RpoB3595 nizingTo determinethe rates ofwhetherthe tworpoB8proteins.suppresses the termination defect of rho), \TP'I - we transduced both rho) and rpoB8 into X X8605, a strain designed to monitor rho-dependent termina- .% Tinre (miar 5 -,75 " lz Ob`.C) .7 tion (21). In this strain, the extent of rho-dependent termi- Lmne 4 nation is indicated by the level of _ *. ..-; 6 P-galactosidase expression * _ *- ;< from a promoterless lac operon fused immediately down- I& = = s w stream from the rho-dependent terminators at the 3' end of _, the trp operon. Introduction of rho) to X8605 resulted in a W.: -If5-fold increase in P-galactosidase expression, confirming the i-! termination defect of this rho allele. This defect in termina- A I, tion is significantly reversed by the introduction of rpoB8. The rpoB8rhol double mutant expresses P-galactosidase at only 30o of the rate of rho) strain (Table 1), indicating that A " Vlow A rpoB8 does suppress the termination defect of rho). To investigate the mechanism of suppression, we turned to A A in vitro experiments. We compared the ability of rhol to terminate transcription at AtR1 with either wild-type or

A :% RpoB8 RNA polymerase as a function of their rates of ox A elongation (Fig. 3). The results of this experiment are in complete accord with the predictions of the kinetic coupling model. At the standard nucleotide concentration (lx), the in vitro assay reproduces the in vivo phenotypes both qualita- tively and quantitatively (Fig. 3, lanes 1 and 2). Rhol shows FIG. 1. Kinetics of transcription elongation from APR by RpoB+ little termination with wild-type RNA polymerase (71% read- and RpoB3595 RNA polymerase. 32]P-labeled RNA samples were through). However, when transcription is carried out by the prepared by transcribing Hinfl-digested pCYC2 with mutant or RpoB8 enzyme, the termination defect is significantly sup- wild-type RNA polymerase for the tii me indicated in a single-round ' transcription assay using either 1 x N1TPs, 2x NTPs, orO.5x N pressed (25% readthrough). The mechanism for suppression The samples were separated by electirophoresis on a 5% polyacryl- appears to reside in tne differences in elongation rates ot tne amide/8 M urea gel. *, Pausing sitess. The positions of transcripts two RNA polymerases. Increasing the rate of elongation of extended to AtR1 site I, II, or III or t(o the end of the template (RO) RpoB8 polymerase so that it approaches that of the wild type are also indicated. eliminates suppression (lanes 3 and 4). Conversely, slowing Downloaded by guest on September 30, 2021 Biochemistry: Jin et al. Proc. Natl. Acad. Sci. USA 89 (1992) 1455 A RpoB+ RpoB8 RpoB3595 [NTPs] 0.25X 0.5X 1X 2X 4X 1X 2X 4X 0.25X 0.5X 1X

Lane 1 2 3 4 5 6 7 8 9 10 1 1

RT-._i_

III a1.

B %Molecules %Molecules %Molecutes RT 6 11 18 28 30 5 9 13 10 18 27 III 1 0 1 3 20 22 24 5 9 16 1 7 21 24 11 26 30 34 29 26 35 34 48 37 35 30 58 46 28 21 20 55 48 23 36 26 1 9

FIG. 2. Termination efficiency of wild-type rho at AtR1 as a function of the elongation rate of RpoB+, RpoB8, and RpoB3595 RNA polymerase. Transcriptions were performed as described and nucleotide concentrations were varied to modulate the rate of elongation of RNA polymerase. The positions of the readthrough transcript (RT) and transcripts terminated at sites I, II, and III are indicated. (A) Relevant portion of the autoradiogram of the transcripts from each reaction. (B) Distribution of each transcript species as percentage of total molecules from a corresponding reaction, obtained by averaging two experiments. Results from the experiments are in close agreement. the rate of wild-type RNA polymerase to that of RpoB8 elongation for RpoB3595 is consistent with the fact that the enables suppression of the termination defect of rhol (lanes concentration of nucleotides in vivo is considerably higher 5 and 6). Taken together, these experiments suggest that, as than the Km ofRNA polymerase for binding nucleotides (33). predicted by the kinetic coupling model, it is simply the altered rate ofelongation ofthe mutant RNA polymerase that permits it to suppress the termination defect of rhol. DISCUSSION Termination Proficiency in Vivo Is Related to the Rate of Kinetic Coupling in rho-Dependent Termination. To ex- Elongation of RNA Polymerase. A final prediction of the plore the idea that termination efficiency at rho-dependent kinetic coupling model is that RpoB8 RNA polymerase, terminators is governed by kinetic coupling between the which exhibits increased termination, should elongate more elongation rate of RNA polymerase and the rate of action of slowly than wild-type RNA polymerase in vivo. Conversely, rho, we characterized the relationship between the altered RpoB3595 RNA polymerase, which exhibits decreased ter- termination properties of mutant RNA polymerases and their mination, should elongate more rapidly than wild-type RNA elongation rates. The in vivo elongation rates of the mutant polymerase in vivo. To test this prediction, we measured the RNA polymerases are inversely related to their in vivo chain growth rate ofRpoB8 and RpoB3595 RNA polymerases termination phenotypes. An overterminating mutant RNA in vivo by a standard assay that follows the rate ofappearance polymerase elongated more slowly than the wild type, while of 13-galactosidase after induction by isopropyl P-D- an underterminating mutant RNA polymerase elongated thiogalactoside (Fig. 4). The justification for and use of this more rapidly than the wild type in vivo. In an in vitro assay to measure elongation rate have been described (31). In

+ cm accord with the predictions ofthe kinetic coupling model, the m m o o RpoB8 RpoB+ lag time for appearance of P-galactosidase was longer in the A OL CIL rpoB8 strain than that of the wild type (Fig. 4A) and was shorter in the rpoB3595 strain than that of the wild type (Fig. [NTPsJ 1X ix 1X 4X IX 0.25X 4B). For rpoB8, the lag was almost twice that ofthe wild type, suggesting that RpoB8 elongates almost twice as slowly in Lane 1 2 3 4 5 6 vivo as wild-type RNA polymerase. This slow rate of elon- gation of rpoB8 may in part explain its increased doubling time (>1.5-fold) compared to the rpoB+ isogenic strain (32). RT o34_ Given the length of f3-galactosidase mRNA, this corresponds to a chain growth rate of 47 nucleotides per sec for wild-type RNA polymerase and 26 nucleotides per sec for RpoB8. For I RpoB3595, there is a much smaller alteration in lag time. In the experiment shown in Fig. 4B, the mutant strain shows an lOo reduction in the lag time, which corresponds to a chain B "MICQ ItIc lels growth rate of 52 nucleotides per sec. Although slight, a 25 60 7 1 48 reduced lag time has been reproducible in four repetitions of RT 7 1 25 this experiment. This small difference in the in vivo rate of III 2 0 3 0 30 22 20 37 11 9 40 40 1 6 9 1 5

Table 1. Effect of rpoB8 on efficiency of termination <1 5 5 2 c1 c1 in a rhol strain FIG. 3. Termination efficiency of mutant rhol at AtRl as a ,B-Galactosidase function of the elongation rate of RpoB+ and RpoB8 RNA polymer- Strain activity, units ase. The experimental conditions were as described in Fig. 2, except X8605 rpoB'rho+ 11 that rhol was used instead of wild-type rho. The positions of the X8605 rpoB'rhol 56 readthrough transcript (RT) and transcripts terminated at sites I, II, X8605 rpoB8rhol 18 and III are indicated. (A) Autoradiogram of the transcription exper- iment. (B) Distribution of each transcript species as percentage of ,1-Galactosidase units are according to Miller (25). total molecules. Downloaded by guest on September 30, 2021 1456 Biochemistry: Jin et al. Proc. Natl. Acad. Sci. USA 89 (1992) A B

MpOB ..5.4% 't a It CP m Q

0 05 LO i5 20 25 30 30 4.0 Time (mim) after IPIG Time (mirn) f IPTG FIG. 4. Initial kinetics of 3-galactosidase (B-gal) induction in rpoB+ and the isogenic rpoB8 mutant (A) or the isogenic rpoB3595 mutant (B). ,3-Galactosidase activity is plotted as (AE)1/2 vs. time of sampling where AE is (-galactosidase at (T = t) - (T = 0). Lines in B were obtained from a least-squares fit of the data points. The lag time for appearance of P-galactosidase is determined by the x-axis intercept of the line for (3-galactosidase activity as discussed by Schlief et al. (30). The mutant and wild-type pair were always measured at the same time to minimize physiological variations. The relative difference in the lag time for appearance of P-galactosidase between the mutant and wild-type strain has been maintained in four independent repetitions of this experiment. The variation in level of(-galactosidase observed between A and B reflects day to day variations in enzyme activity. IPTG, isopropyl /3-D-thiogalactoside. rho-dependent termination assay, the termination profile of termination sites, makes them exquisitely sensitive to small the mutant RNA polymerases was very similar to that ofwild changes in the elongation rate of RNA polymerase and the type, provided that they were compared at equivalent elon- rate of action of rho. Because efficient rho-dependent termi- gation rates. Thus, the altered in vivo termination phenotypes nators are created from individually inefficient termination of the mutants can be explained solely by their altered rates events, a small change in the efficiency of each elementary of elongation, although we cannot eliminate the possibility event will be multiplicative and therefore have a very large that other changes in the mutant enzymes also contribute to effect on overall termination efficiency. the phenotype. Regulatory Function of Kinetic Coupling in Gene Expres- Further evidence for the intimate relationship between the sion. The intimate kinetic coupling between the elongation rate of action of rho and the rate of elongation of RNA rate of RNA polymerase and the rate of action of rho could polymerase comes from our analysis of the mechanism by enable the cell to govern termination efficiency at rho- which rpoB8 suppresses the termination defect ofrho). Rhol dependent terminators. Clearly, the extent of termination at acts more slowly in vitro and leads to almost complete an individual terminator can be modulated by altering the readthrough of rho-dependent terminators in vivo. A mutant nucleotide sequence or structure so that it encodes more or RNA polymerase that elongates slowly in vivo significantly less of a pause for RNA polymerase. suppresses the termination defect of rhol. That the altered From a regulatory point of view, a much more exciting elongation rate ofthe mutant RNA polymerase is responsible prospect is the possibility that proteins or other factors for suppression was indicated by in vitro studies showing that modulating termination and antitermination do so by altering slowing down the wild-type RNA polymerase to mutant rates the elongation kinetics of RNA polymerase. Our studies on also results in suppressing the termination defect. the in vitro properties of RpoB8 RNA polymerase have Our experiments demonstrate directly that termination indicated that increasing the Km ofRNA polymerase not only efficiency at rho-dependent terminators is governed by a decreases the rate of addition of nucleotides but also in- precise concurrence between the rates of action of RNA creases the time spent at many pause sites (19). Because the polymerase and rho. Very small increases in the rate of behavior ofthe enzyme at pause sites is sensitive to the same elongation of RNA polymerase or decreases in the rate of parameters affecting nucleotide addition, factors decreasing action of rho lead to significant readthrough at many rho- the elongation rate would not only decrease the rate of dependent terminators. Conversely, a small decrease in the addition of nucleotides but also enhance termination by elongation rate of RNA polymerase significantly increases increasing pausing. Conversely, factors increasing the chain termination with wild-type rho. The narrow range over which growth rate of RNA polymerase would decrease pausing and the rates of RNA polymerase and rho are matched for cause antitermination. effective termination is shown most graphically by the func- There is some evidence that bacteriophage A antitermina- tional interaction between rpoB8 and rho). Rhol works tion proteins may work in part by increasing the elongation sufficiently slowly that it is quite defective in terminating rate ofRNA polymerase so that rho and RNA polymerase are transcription by wild-type RNA polymerase. A <2-fold de- no longer synchronized for effective termination. The AQ crease in the rate ofelongation ofRNA polymerase, as is seen protein, which promotes antitermination, has been shown to in vivo with RpoB8, results in almost complete suppression reduce pausing (17). Likewise, the AN protein, another of the termination defect by resynchronizing the two pro- antiterminator, also reduces pausing (W. Whalen, A. Das, teins. Some of the "super rho" or rhos mutations, which and J. Greenblatt, personal communications). In this regard, enhance termination and shift the pattern of termination so it is conceivable that a class of the nusD rho mutants, which that it occurs predominantly at the proximal rho termination prevent the A antitermination system from working (36), sites (34, 35), may do so by increasing the rate of action of increases the rate of action of rho. This would restore rho, thus perturbing kinetic coupling. termination at rho-dependent terminators by resynchronizing Interestingly, the very structure of rho-dependent termi- the rate of action of rho to the more rapid rate of elongation nators, consisting of multiple, closely spaced inefficient exhibited by N-modified RNA polymerase. Interestingly, the Downloaded by guest on September 30, 2021 Biochemistry: Jin et al. Proc. Natl. Acad. Sci. USA 89 (1992) 1457 in vivo transcription rate of rRNA operons is faster than that the use of kinetic mechanisms to coordinate and regulate of mRNA operons (37). Since rRNA operons are subject to cellular processes. the rRNA antitermination system (38), it is possible that this rRNA antitermination system also works in part by altering 1. Platt, T. (1986) Annu. Rev. Biochem. 55, 339-372. 2. Yager, T. D. & von Hippel, P. H. (1987) in Escherichia coli and Salmo- the elongation kinetics of RNA polymerase. nella typhimurium: Cellular and Molecular Biology, eds. Neidhardt, There is evidence that the elongation rate of RNA poly- F. C., Ingraham, J. L., Low, K. B., Magasanik, B., Schechter, M. & merase also affects termination at intrinsic terminators. The Umbarger, H. E. (Am. Soc. Microbiol., Washington), pp. 1241-1275. 3. Richardson, J. P. (1990) Biochim. Biophys. Acta 1048, 127-138. RpoB8 enzyme shows increased termination at some intrinsic 4. Morgan, E. A., Bear, D. G. & von Hippel, P. H. (1984) J. Biol. Chem. terminators, as well as at rho-dependent terminators both in 259, 8664-8671. vivo and in vitro. Likewise, rpoB3595 also shows decreased 5. Ceruzzi, M., Bektesh, S. L. & Richardson, J. P. (1985) J. Biol. Chem. 260, 9412-9418. termination at some intrinsic terminators. In vitro studies 6. Lau, L. F., Roberts, J. W. & Wu, R. (1982) Proc. Natl. Acad. Sci. USA indicating that nucleotide concentration affects termination 79, 6171-6175. efficiency at some intrinsic terminators are also in accord 7. Lau, L. F., Roberts, J. W. & Wu, R. (1983) J. Biol. Chem. 258, 9391-9397. with this idea (39-41). The coupling of elongation rate and 8. Morgan, W. D., Bear, D. G. & von Hippel, P. H. (1983) J. Biol. Chem. termination efficiency may simply be explained by kinetic 258, 9553-9564. competition between elongation and termination at intrinsic 9. Morgan, W. D., Bear, D. G. & von Hippel, P. H. (1983) J. Biol. Chem. 258, 9565-9574. terminators. A slower rate of incorporating nucleotides 10. Lowery-Goldhammer, C. & Richardson, J. P. (1974) Proc. Natl. Acad. would be kinetically favorable to termination and vice versa. Sci. USA 71, 2003-2007. Alternatively, ifa conformational change in RNA polymerase 11. Brennan, C. A., Dombroski, A. J. & Platt, T. (1987) Cell 48, 945-952. 12. Schmidt, M. C. & Chamberlin, M. J. (1984) J. Biol. Chem. 259, 15000- is a prerequisite for establishment of the termination mode at 15002. intrinsic terminators, the rate ofelongation may influence the 13. Das, A., Merril, C. & Adhya, S. (1978) Proc. Natl. Acad. Sci. USA 75, fraction of RNA polymerases in the termination-competent 4828-4832. 14. Guarente, L. P. (1979) J. Mol. Biol. 129, 295-304. conformation. Thus, the enhanced elongation rate of RNA 15. Jin, D. J. & Gross, C. A. (1989) Mol. Gen. Genet. 216, 269-275. polymerase modified by the AN and AQ antitermination 16. Greenblatt, J., McLimont, M. & Hanly, S. (1981) Nature (London) 292, systems invoked to explain antitermination at rho-dependent 215-220. 17. Yang, X. & Roberts, J. W. (1989) Proc. Natl. Acad. Sci. USA 86, terminators may also explain why these systems antitermi- 5301-5305. nate at intrinsic terminators as well. 18. Fisher, R. F. & Yanofsky, C. (1983) J. Biol. Chem. 258, 8146-8150. Recently, quantitative thermodynamic and kinetic models 19. Jin, D. J. & Gross, C. A. (1991) J. Biol. Chem. 266, 14478-14485. have been developed to account for the positions and effi- 20. Jin, D. J., Walter, W. & Gross, C. A. (1988) J. Mol. Biol. 202, 245-263. 21. Guarente, L. P. & Beckwith, J. (1978) Proc. Nail. Acad. Sci. USA 75, ciencies of the intrinsic terminators of E. coli (42, 43). Using 294-297. transition state theory, these authors have shown that the 22. Jin, D. J. & Gross, C. A. (1988) J. Mol. Biol. 202, 45-58. activation free energy barriers to elongation and to termina- 23. Hager, D. A., Jin, D. J. & Burgess, R. (1990) Biochemistry 29, 7890- 7894. tion are of comparable heights at intrinsic termination sites 24. Finger, L. R. & Richardson, J. P. (1981) Biochemistry 20, 1640-1645. and, by analogy, at factor-dependent termination sites as 25. Miller, J. H. (1972) Experiments in Molecular Genetics (Cold Spring well. As a consequence, observed termination efficiencies at Harbor Lab., Cold Spring Harbor, NY). such sites can easily be modulated by small changes in the 26. Rosenberg, M., Court, D., Shimatake, H., Brady, C. & Wulff, D. L. (1978) Nature (London) 272, 414-423. relative rates of transcript elongation and release. An RNA 27. Court, D., Brady, C., Rosenberg, M., Wulff, D. L., Behr, M., Mahoney, polymerase characterized by a slower elongation rate would M. & Izumi, S. (1980) J. Mol. Biol. 138, 231-254. thus be expected to exhibit increased termination efficien- 28. Beckwith, J. R. (1963) Biochim. Biophys. Acta 76, 162-164. 29. Richardson, J. P. & Carey, J. L. I. (1982) J. Biol. Chem. 257, 5767-5771. cies, and vice versa, and for well-characterized cases the 30. Schlief, R., Hess, W., Finkelstein, S. & Ellis, D. (1973) J. Bacteriol. 115, changes in termination efficiencies that follow from such rate 9-14. differences can be calculated. Our experimental results agree 31. Kepes, A. (1969) Prog. Biophys. Mol. Biol. 19, 199-236. with these theoretical expectations. 32. Jin, D. J. & Gross, C. (1989) J. Bacteriol. 171, 5229-5231. 33. Kingston, R. E., Nierman, W. C. & Chamberlin, M. J. (1981) J. Biol. Kinetic mechanisms have been proposed to underlie the Chem. 256, 2787-2797. fidelity of DNA replication, the vectorial nature of secretion, 34. Tsurushita, N., Shigesada, K. & Imai, M. (1989) J. Mol. Biol. 210, 23-37. and the attenuation response. The high fidelity of DNA 35. Mori, H., Imai, M. & Shigesada, K. (1989) J. Mol. Biol. 210, 39-49. polymerase I derives, in part, from exonucleolytic editing. 36. Das, A., Gottesman, M. E., Wardwell, J., Trisler, P. & Gottesman, S. (1983) Proc. Natl. Acad. Sci. USA 80, 5530-5534. Surprisingly, base discrimination is not the primary determi- 37. Bremer, H. & Dennis, P. P. (1987) in Escherichia coli and Salmonella nant ofediting specificity. Instead, the editing contribution to typhimurium: Cellular and Molecular Biology, eds. Neidhardt, F. C., the fidelity of DNA polymerase I comes primarily from a Ingraham, J. L., Low, K. B., Magasanik, B., Schechter, M. & Um- kinetic mechanism in which continued DNA chain elongation barger, H. E. (Am. Soc. Microbiol., Washington), pp. 1527-1542. from a mismatched base is the exonu- 38. Morgan, E. A. (1986) J. Bacteriol. 168, 1-5. very slow, allowing 39. Stroynowski, I., Kuroda, M. & YanofskypC. (1983) Proc. Natl. Acad. clease more time to work (44). Likewise, it has been sug- Sci. USA 80, 2206-2210. gested that some GTP-binding proteins in the secretory 40. Landick, R. & Yanofsky, C. (1984) J. Biol. Chem. 259, 11550-11555. pathway function by coupling the slow hydrolysis of GTP to 41. Reynolds, R., Bermudez-Cruz, R. M. & Chamberlin, M. J. (1991)J. Mol. Biol. 223, in press. an interaction between vesicles and acceptor molecules, 42. Yager, T. D. & von Hippel, P. H. (1991) Biochemistry 30, 1097-1118. ensuring vectorial transport by a kinetic proofreading mech- 43. von Hippel, P. H. & Yager, T. D. (1991) Proc. Natl. Acad. Sci. USA 88, anism (45). Finally, attenuation mechanisms that couple the 2307-2311. availability of an amino acid to transcription of genes encod- 44. Kuchta, R. D., Benkovic, P. & Benovic, S. J. (1988) Biochemistry 27, 6716-6725. ing the biosynthetic enzymes synthesizing this amino acid 45. Baker, D., Wuestehube, L., Schekman, R., Botstein, D. & Segev, N. rely on the relative rates of movement ofribosomes and RNA (1990) Proc. Nail. Acad. Sci. USA 87, 355-359. polymerase (46). Our work provides an additional example of 46. Yanofsky, C. (1988) J. Biol. Chem. 263, 609-612. Downloaded by guest on September 30, 2021