Proc. Nati. Acad. Sci. USA Vol. 88, pp. 6018-6022, July 1991 Biochemistry A /8 subunit mutation disrupting the catalytic function of RNA polymerase (/ clearance/pausing/elongation rate) JOOKYUNG LEE*, MIKHAIL KASHLEVt, SERGEI BORUKHOV*, AND ALEX GOLDFARB*t *Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, NY 10032; and tInstitute of Molecular Genetics, U.S.S.R. Academy of Sciences, Moscow, U.S.S.R. Communicated by E. Peter Geiduschek, March 25, 1991 (received for review February 13, 1991)

ABSTRACT The substitution of the evolutionarily con- Fl, F2, F3, or F4. The recipient plasmids pMKA351, served Glu-813 for lysine in the (3 subunit of RNA polymerase pMKA335, or pMKA312 were digested with the same endo- (RNAP) causes a partial loss of function in the assembled nucleases completely or (for Pst I) partially to generate RNAP. In the presence of the four ribonucleoside triphos- appropriate targets for the insertion of each fragment. The phates, the mutant RNAP displayed a decreased frequency of donor and the recipient digests were ligated, digested with promoter clearance and diminished elongation rate. Both HindIII and BamHI to eliminate the parental plasmids, and defects could be compensated by raising the ribonucleoside transformed into the HB10l[pLacIQI host (14). For fragments triphosphate concentration. In the abortive initiation reaction F1, F2, and F3, successful fragment exchange was confirmed limited by the incomplete set of ribonucleoside triphosphates, by the appearance ofinducible full-sized polypeptide due to the mutant RNAP generated aberrant patterns of products the replacement of the BamHI frameshifting. linker of the indicative of their enhanced loss from the RNAP-promoter recipient plasmid. To determine the genetic makeup of the complex. A model is proposed, attributing the multiple effect recombinant plasmids the transformants were plated with of the mutation to the malfunctioning of the RNAP active and without Rif in the absence and in the presence of center. isopropyl fJ-D-thiogalactoside. For sequence determination, the fragment F2 (Fig. 1A) was inserted in both orientations RNA polymerase (RNAP) of Escherichia coli is a multifunc- into the Pst I site of the multiple cloning site of the Ml3mpl9 tional multisubunit enzyme performing principal reactions of vector. The directly oriented M13 insert was sequenced in the expression (1, 2). A key step ofits functional cycle is the upstream direction from the distal Pst I site. The inversely transition from the initial transcribing complex (ITC) to the oriented M13 insert was shortened by generating the Hpa elongating complex (EC) (3), also called "promoter clear- I/Sma I deletion using the Sma I site within the vector ance. " In the ITC, the a2,p3'c holoenzyme is stably anchored sequence. The resulting clone was sequenced in the down- at the promoter, continuously making and releasing nested stream direction from the Hpa I site in rpoB. RNA oligomers, up to 9 (nt) in length, in repeated Biochemical Techniques. RNAP was reassembled from acts of abortive initiation (3-7). Promoter clearance involves individually overexpressed subunits as described (14, 15). the release of the o factor, relinquishing of the anchoring The reassembled enzymes were titrated in a reaction (see contacts, stabilization of the RNA product in the ternary below) on the T7 Al promoter by measuring the formation of complex, and commencement of processive elongation by the abortive product CpApU from CpA (here and later the core enzyme a2f313' (3, 7-9). The propagation of EC along boldface type designates the priming ) and the template occurs in rapid bursts punctuated by "pausing" [a-32P]UTP, to determine the point of saturation of the at specific sites (10-13). Abortive initiation and pausing are template with the enzyme. reactions were probably linked to the catalytic mechanism ofRNAP because performed in 10 ,ul of the reaction buffer (14) containing -0.1 their kinetic parameters are influenced by the concentration pmol of the 130-base-pair-long DNA fragment carrying the of ribonucleoside triphosphates (rNTP). phage T7 Al promoter (a gift from H. Heumann, ref. 17) and We have developed a method of probing RNAP vital a saturating amount (0.3-0.4 pmol) of reconstituted RNAP. features with transdominant mutations that lead to partial RNAP was preincubated with DNA in the presence of 0.5 loss offunction (14, 15). Here we characterize a single amino mM of the priming dinucleotide (CpA or ApU obtained from acid substitution in the p subunit, Glu-813 -- Lys (E813K), Sigma) for 15 min at 37TC, and the reactions were started by which simultaneously accelerates oligomer loss at the ITC adding the substrate mixture. The composition and the final step, enhances pausing at the EC step, and decreases the concentrations of rNTP as well as the source of radioactivity frequency of ITC/EC transition, apparently through the are indicated in figure legends. In some reactions, terminating disruption of the enzyme's active center. 3'-deoxynucleoside triphosphate (Pharmacia) was included, as indicated. The reactions were allowed to continue for 15 MATERIALS AND METHODS min (unless indicated otherwise), stopped by adding ethyl- enediaminetetraacetate to the concentration of 50 mM, and Genetic Techniques. The plasmids pXT7,3, its rpoB1855- the mixtures were applied directly to polyacrylamide/8 M carrying derivative pJL97 (16), and pMKA92 (14) were urea gels (20% acrylamide/3% NN'-methylenebisacryla- described. pMKA351, pMKA335, and pMKA312 were iso- mide), which were run and autoradiographed as described lated in the course of linker-insertion mutagenesis of (14). The sequences of abortive were deter- pMKA92 (M.K., unpublished observations). For mapping of mined by using UTP, CTP, GTP, and ATP as alternative the mutation (Fig. LA), donor pJL97 DNA was digested with appropriate restriction endonucleases to generate fragments Abbreviations: EC, elongating complex; rNTP, ribonucleoside tri- phosphate(s); ITC, initial transcribing complex; Rif, ; The publication costs of this article were defrayed in part by page charge RNAP, RNA polymerase; nt, nucleotide(s); E813K, Glu-813 -+ Lys; payment. This article must therefore be hereby marked "advertisement" RNAP813, RNAP carrying E813K; RNAPwT, wild-type RNAP. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 6018 Downloaded by guest on September 29, 2021 Biochemistry: Lee et al. Proc. Natl. Acad. Sci. USA 88 (1991) 6019

(81 3) A RpoB 1855 (1287)(1319) (2?37) (433) (736) (965) P S p C H P 1 (531) pJL97 PT7 -I--

Hindill C H FIG. 1. rpoB mutation E813K (rpoB1855). p Z mI F1 p (A) Localization of rpoB855 by transfer of the Plac F2 fragments F1, F2, F3, and F4 from plasmid pMKA351 - & 1I_ pJL97 to one of the recipient plasmids of the (516) t pMKA series. Each recipient plasmid carries a BamHl BamHI linker insertion (upward arrows) that (609) H S inactivates f3 subunit function. For pMKA351 and pMKA335, the insertion causes the reading- Plac I--X -' T F 3 frame shift (hatched areas). X and R designate pMKA335 - = = R(1 ZIZF the rpoB1855 and a rifampicin (Rif)-resistance (51 6) allele, respectively. PT7 and Plac symbolize pro- BamHlt moters. Restriction sites (downward arrows) are (850) P P indicated as follows: P, Pst I; C, Cla I; H, Hpa Plac F4 I; and S, Sma I. Codon numbers are given in pMKA312 -~ ~ III }- parentheses. The failure ofrecombinant plasmids (516) to confer isopropyl f3-D-thiogalactoside-depen- BamHl dent Rif resistance upon the host was taken as (1187) evidence that the transferred fragment carried B the lethal rpoB1855 mutation. (B) Sequence data E813K and homology alignment. Nucleotide sequences around codon 813 are shown in the box with the TTC a -kAC3- mutant base set in lowercase. Amino acid se- quences (from top to bottom) are from the 83 T 'TCt.aSAAGGAC subunit ofE. coli and from homologous segments TacaT ofPseudomonas putida, the chloroplasts of spin- ach and liverwort, Saccharomyces cerevisiae, WNGYNF DSTILVS:N, (820) .... (875) A E V TG' GCI VGKV T a K and Drosophila melanogaster. Codon numbers 4 N G F mN F E S I L S (825) .... (881-) A N V GA G DS ILVGrNKV T P s are given in parentheses. Positions of identity to rN _ ntvFD A V L I S E (675--) .... (730) S W V ETT G D LVV G KL P WEGYN.,'SEDAIL.12'-NE (670).... (725) S W V E T G E V NT V K L T Q the E. coli sequence are screened. The putative I 7 (643) .... (903) V R V S G E D V I I C K T T P I nucleotide-binding motif (see Discussion) is Y IN, E D S M Mv NA Q I T Y SY N Q E D S V I - N A (747) . . (I87)1 R V S G D D V 'VIG K T ' Lshown underneath. These amino acid sequences N-x-x-D G-x-x-x-x-G-K are referenced elsewhere (14). sources of the a-32p label and by using terminating 3'- plasmid-encoded f3, the mutation was mapped within the deoxyribonucleoside triphosphates. Transcripts were quan- 697-bp overlap of fragments F2 and F3, between Hap I and titated by cutting bands out of the gel slab and measuring the Pst I restriction sites. This segment was sequenced in both Cherenkov radioactivity. orientations, and the mutation was found to be the substitu- To make synchronized stalled +20 complexes with RNAP tion of Glu-813 for lysine (Fig. 1B, box). carrying E813K (RNAP813), the ApU-primed reaction was E813K Inhibits Promoter Clearance. RNAP813 holoenzyme continued for 30 min with ATP and GTP (100 AuM each) and carrying p subunit with the substitution was reconstituted 5 AM of [a-32P]CTP. With wild-type RNAP (RNAPwT), the from individually overexpressed subunits (14, 15). As the ApU-primed reaction was carried out for 15 min with ATP, control, RNAP'" carrying the (3 subunit specified by the GTP, and [a-32P]CTP (5 ,uM each). The reaction mixtures parent plasmid pXT7I3 was assembled. Both preparations were then supplemented with RNase-free bovine serum contained nearly stoichiometric amounts of a. However, albumin (Pharmacia) at 250 Ag/ml and applied onto an FPLC RNAP813 was totally inactive in vitro on T4 DNA as well as Fast Desalting column (Pharmacia) equilibrated with 40 mM on several promoter DNA fragments, whereas RNAPWT Tris, pH 7.9/60 mM NaCl. The UV-absorbing peak fraction displayed high specific activity. Yet, in all cases, RNAP813 was collected and stored at +40C. To resume elongation, the generated abortive oligomers with high efficiency (data not samples were warmed to 370C and supplemented with the shown). Thus, RNAP813 is qualitatively deficient in the missing components of the standard reaction containing promoter clearance step of the transcription cycle. either 5 ,RM or 50 ,uM ofeach ofthe four rNTP. Aliquots were Fig. 2 presents the quantitation of this defect for the phage withdrawn at time intervals, and then the reactions were T7 Al promoter. To limit transcription to a single round, the stopped and analyzed in a 15% (acrylamide/N,N'- following scheme was used. The initial transcribed sequence methylenebisacrylamide, 14:1) gel, as described above. of this promoter is ApUpCpGpA--, and the next uridine is located at position +21. The transcription was initiated with the dinucleotide ApU, whereas UTP was substituted with its RESULTS chain-terminating analog 3'-deoxyuridine triphosphate. Un- Mapping of rpoBl855. The mutation rpoBl855 was orig- der these conditions, EC is expected to propagate unimpeded inally isolated as a dominant lethal mutation, after random until it stalls at position +21. Because each template fragment mutagenesis ofthe expression plasmid pXT7,3, which carries can accommodate only one stalled EC, the maximum number a Rif-resistant allele of rpoB (16). To map rpoB1855, a series ofstalled complexes formed at RNAP excess should be equal of in vitro exchanges of DNA fragments (Fl, F2, F3, and F4) to the number of template molecules. The time needed to was performed between plasmids as illustrated in Fig. lA. reach this maximum should reflect the rate of promoter Using Rif resistance as the indicator of functionality of clearance. Downloaded by guest on September 29, 2021 6020 Biochemistry: Lee et al. Proc. Natl. Acad. Sci. USA 88 (1991) A *Why E 81 3 K B 65-nt runofftranscript by adding large excess of natural UTP (data not shown). The two unexpected bands (X and Y in Fig. 2A) are attributed to artifacts. X represents a start site other than the Al promoter, as was demonstrated by transcript .53 3 mapping with restriction enzymes (data not shown). Y, which -0 accumulated only under the conditions of arrested elonga- 2 ,---- ',i1*4|W * tion, apparently represents the activity of secondary RNAP _z~~VWt molecules binding at the vacated Al promoter after the initial x 2 complexes have moved to position +27 and/or +33. x E Quantitation ofcumulative radioactivity in bands 21 and 27 C) of Fig. 2A is shown in Fig. 2B. Clearly, the accumulation of

~0 1 stalled EC in the RNAPr reaction (open symbols) was = essentially complete between 0.5 and 1 min. For RNAP813 0~~~~~~~ (closed symbols), the number of stalled EC approached a plateau only by 30 min. The level of this plateau was

o_NNo CM E reproducibly at least three-quarters that of RNAP"", indi- 5D .. C a -_ C 10 20 30 cating that most available template fragments were eventu- C:)- o~rn o Ou u u m i n u t e s ally used by the RNAP813. It should be noted that RNAP813 generated much more abortive oligomers per productive c WT E813K D initiation than RNAPI (Fig. 2A). The use ofthe promoter by RNAP813 was not only slow but required unusually high concentrations of rNTP (Fig. 2C). At rNTP concentrations -~ -~ 33 below 25 ,uM, few stalled EC were generated by RNAP813, ___a*bob~b _ - 27 even after prolonged incubation, whereas RNAPWT used the - 21 cr) promoter with high efficiency in the rNTP concentration range from 5 to 100 ,uM. The slow accumulation of EC can, in principal, be due to either premature dissociation of RNAP813 from the promoter E or decreased rate of ITC -* EC transition. The latter possi- 0. bility seems more likely because of the effect of substrate N_*_T - Y concentration as well as the relative overproduction of abor- tive oligomers by RNAP813. This explanation is reinforced by Q~~ **ROi 43 2 the experiment of Fig. 2D, in which the mutant catalytic ITC * 'NI i. was challenged with DNA competitor poly[dA-dT] as it was _~O~)O_ - (N 10 15 20 producing the abortive tetramer CpApUpC. In this experi- m i n u t e s ment, the -1,+1 primer CpA had to be used because ApU is complementary to poly[dA-dT]. The formation of chains longer than CpApUpC was prohibited because GTP and ATP FIG. 2. Analysis of promoter clearance. (A) Time course c f a single-round transcription reaction. RNA chains were initiated vvith 1 2 3 4 5 6 7 8 9 1 o 11 1 2 1 4 5 r, 1 ApU in the presence of ATP and GTP (0.1 mM each), 50 /M [a-32P]CTP, and 0.5 mM of 3'-deoxyuridine triphosphate, at 3-Ifold 65 molar excess of RNAP over template, and the reactions w,ere stopped after indicated time intervals. Numbers 21, 27, 33, and1 45 indicate length in nucleotides ofthe transcripts arrested within the Al i~~~li transcription unit. Unidentified abortive products are resolved at the 'us w~~w~.Iu~~.sI bottom of the gel. Products X and Y are due to artifacts discussedd in .i the text. (B) Quantitation ofarrested EC. Cumulative radioactivit'y of bands 21 and 27 ofA, expressed as modified cmp after correctionifor the number of labeled phosphates. Each point was obtained by adding the Cherenkov count of band 21 to two-thirds ofthe counit of band 27 to account for the fact that the two transcripts contain 4;and 6 radioactive cytosine nucleotides, respectively. WT, open symboIs; E813K, closed symbols. (C) Dependence of promoter clearance:on substrate concentration in a single-round reaction. The ApU-prinned reactions similar to those in A were incubated for 15 min with 0.5imM of triphosphate and the indicated concentrations of 1lml ,-..--1 3'-deoxyuridine E.WS_ ATP, GTP, and [a-32P]CTP, maintained at constant specific raidio- 4AML 26- 0 *0~S: activity. (D) Stability of E813K ITC. Time course of productioin of 20- * CpApUpC in the CpA-primed reaction with 100 ,uM UTP and 50 IM to r 10 [a-32P]CTP. Two parallel reactions were allowed proceed foi Lf U) U) :)Un)Lr) min, after which poly[dA-dT] was added to one of them (open min:o c Ln CD °o Lcm -- ° symbols) to the final concentration of 0.1 mg/ml. A control demion- strating instantaneous inactivation offree RNAP by poly[dA-dT] iwas performed (data not shown). E813K WT (50IM NTP) (5WM NTP 50uM NTP. When this experiment was done (Fig. 2A), it was noted tLhat the EC stalled not at one but at several points correspondling FIG. 3. Analysis of elongation. The EC complexes stalled at positions +20 and +26 (lanes 0) were purified from unincorporated to uridine positions +21, +27, +33, and +45. We attribute rNTP by gel filtration. They were incubated with either 50 ,M (lanes this read-through phenomenon to pyrophosphorolysis;and 1-6, 13-18) or 5 jAM (lanes 7-12) of nonradioactive rNTP for the~presence of contaminating UTP in the commercial lot:s of indicated time periods under otherwise standard conditions. Position other rNTP (18). The stalled EC could be chased to yield the of the 65-nt runoff transcript is indicated. Downloaded by guest on September 29, 2021 Biochemistry: Lee et al. Proc. Natl. Acad. Sci. USA 88 (1991) 6021 were absent. It can be seen that after the addition of the promoters. The assays were done in the absence of DNA competitor the catalytic reaction slowed down with an ap- competitors, allowing an average mutant RNAP to dissociate parent half-life of -10 min. A similar level of poly[dA-dT] and rebind once per 15 min ofthe steady-state reaction (based resistance was seen during catalytic pentamer (CpAp- on the 10-min complex half-life, see Fig. 2D). Fig. 4C UpCpG) formation. Thus, although the mutant ITC is less presents the control multiple-round reaction containing the stable than the wild-type complex that was totally poly[dA- four natural rNTP, in which promoter clearance was allowed dT] resistant (data not shown), ITC dissociation cannot to occur. account for the dramatic inhibition of the initiation rate In the " +4" reaction (Fig. 4A), the pentamer CpApUpCpG observed, especially at suboptimal substrate concentrations. was the predominant product of RNAP"'. By contrast, We conclude that the principal deficiency of RNAP813 is in RNAP813 also generated CpApU in substantial amounts. the ITC/EC transition. Furthermore, the total production of oligomers by RNAP813 E813K Slows Elongation. We next compared the elongation was -three times higher than by RNAPWT. This result could rate (and its dependence on rNTP concentration) of the not reflect a larger number of active complexes because the E813K and wild-type EC that were synchronized at position number of available promoters was the limiting factor. More- +20 by omitting UTP from the initial reaction mixture and over, as was shown above (see Fig. 2 A and B), the number of productive complexes was in fact slightly lower in the then purified by gel filtration (Fig. 3). When the isolated RNAP813 than in the wild-type reaction. Thus, the enhanced stalled EC (lanes 1, 7, and 13) were allowed to resume productivity of RNAP813 can be explained only by faster elongation by the addition of the four standard rNTP, a catalytic rate of the mutant complexes. dramatic difference between RNAP813 (lanes 1-6) and In the " +5" reaction (Fig. 4B), the higher overall initiation RNAPWT (lanes 7-18) was observed. With RNAP813 almost rate and the generation of trimer CpApU by RNAP813 were no chain extension was evident at 5 puM of rNTP (data not again evident. However, the hexamer CpApUpCpGpA was shown), whereas it took 10 times higher concentration of dramatically underproduced by RNAP813. The production of rNTP (50 ,M, lanes 1-6) to achieve the elongation rate and CpApUpCpGpA by RNAP813 could be brought to the wild- the pausing pattern seen at 5 AtM rNTP with RNAPWT (lanes type level by increasing the ATP concentration -10-fold 7-12). Under the same rNTP concentration (50 ,.M rNTP), (compare 50 ,uM WT and 500 ,uM E813K lanes). Under these RNAPWT and RNAP813 synthesized chains of comparable conditions, production of the pentamer CpApUpCpG by length after 30 sec (lane 14) and 10 min (lane 6), respectively. RNAP813 was at least three times higher than by RNAPWT. Aberrant Abortive Initiation by RNAP813. That the pro- Thus, the efficiency of extension of CpApUpCpG to CpAp- moter clearance and elongation defects of RNAP813 could be UpCpGpA is inhibited by the mutation '30-fold. In conclu- compensated by excess rNTP suggests that the mutation may sion, the abortive initiation reaction performed by RNAP813 have affected the catalytic function of the enzyme. To reveals two distinct characteristics ofthe mutant ITC: afaster explore this possibility, we analyzed in more detail abortive catalytic rate with regard to the shorter abortive products and initiation (Fig. 4 A and B). In this experiment, RNA chains a strong barrier in extending RNA chains between the +4G were primed with CpA corresponding to the initial tran- and +5A positions of the initial transcribed sequence. scribed sequence CpApUpCpGpApG- . To prevent pro- moter clearance, 3'-deoxy analogs of GTP (A) and ATP (B), which terminate chains at positions +4 and +5, respectively, DISCUSSION were added. RNAP was in excess, thus limiting the maximum The substitution E813K causes multiple functional changes in number of catalytic complexes to the number of available RNAP. RNAP813 was found to have (i) a qualitatively different A B C CpA-0.5 mM CpA-0.5 mM CpA-0.5 mM ^lJTPF CTP-0.1 mM *UTP. CTP, GTP-0.1 mM *UTP, CTP. GTP-0.1 mM WI- E813K WT E813K WT E813K ; L4 - Runoff

CpApUpCpGpA

% do , -CpAppUpCpG ~U -CpApU

L') 0 0 0 0 I c) 0 0 0D IC 00C00L)DO O O uI)0 0O Iooo00 o \ Ln 0 IC 0 N IC 0 Lo 0 C(' LO 0 LC) 0 N Lr) 0 IC) 0 c LnoC0 o CN 0 IC 0 - - - Nc - N S - ct Lf)- C>J LO) C\J Ln N Lr) it M ATP . 13-deoxy GTP .L M 3-deoxy ATP

FIG. 4. Analysis of abortive initiation. Comparison of the RNAPWr and RNAP813 in the standard in vitro assay with (A and B) or without (C) chain-terminating nucleotides. 3'-deoxy GTP, 3'-deoxyguanine triphosphate; 3'-deoxy ATP, 3'-deoxyadenosine triphosphate. Downloaded by guest on September 29, 2021 6022 Biochemistry: Lee et al. Proc. Natl. Acad. Sci. USA 88 (1991) pattern of abortive products, (ii) a diminished frequency of the are pertinent to the role of the Glu-813 residue in the RNAP ITC -+ EC transition, and (iii) an enhanced pausing. The molecule. It is tempting to speculate that Glu-813 directly decrease in promoter clearance (30 min vs. 30 sec in the contacts the RNA product in the active center. In this single-round reaction of Fig. 2 A and B) and the elongation connection, we note that Glu-813 is a member of a conserved slowdown were sufficient to bring transcription in the multiple- bipartite nucleotide binding motifN-x-x-D. . .G-x-x-x-x-G-K round reaction to almost a total halt (Fig. 4C). (Fig. 1B) which is found in many GTP- and ATP-binding The effect on promoter clearance and the apparent pro- (20). longation of pausing can be compensated by higher rNTP So far chemical and genetic data have implicated other concentration. Hence, both defects could be from a de- localities of the 83 polypeptide in the catalytic function. creased affinity for the elongation substrate. This explana- Crosslinking experiments (21) and site-directed mutagenesis tion, however, does not fit the observed changes in the (14) have positioned Lys-1065 and His-1237 in close vicinity abortive reaction. For example, the enhanced production of to the priming nucleotide. RNAP from the Rif-resistant CpApUpCpG and the formation of CpApU by RNAP813 (Fig. mutant rpoB8 (Gln-513 -* Pro) revealed a changed Km for 4A) cannot be explained by a reduced binding of rNTP. purine nucleotides during elongation (22). Systematic muta- Similarly, RNAP813 and RNAPWT generated CpApUp- tional analysis ofthese localities will help to clarify their roles CpGpA with the same rate at 500 uM and 50 ,uM ATP, and relationship to each other. respectively (Fig. 4B). If the substrate binding were the only defect, under these conditions the two enzymes should have We are grateful to Katya Zalenskaya for advice on RNAP recon- produced the shorter oligomers at the same rate. In reality, stitution and to Vadim Nikiforov, Michael J. Chamberlin, and RNAP813 dramatically overproduced CpApUpCpG and George Kassavetis for discussion and criticism. This work was CpApU, which contradicts the notion of a decreased affinity supported by grants from the National Institutes of Health for rNTP. (GM30717) and the American Cancer Society (MV-285). M.K. was We propose that the primary effect of the mutation is a a recipient ofthe Soros Foundation-Soviet Union Travel Fellowship. destabilization of the nascent RNA product in the active center. Assuming that the rate of abortive initiation is limited 1. von Hippel, P. H., Bear, D. G., Morgan, W. D. & McSwiggen, by the release of CpApUpCpG, the enhanced loss of this J. A. (1984) Annu. Rev. Biochem. 53, 389-446. product from the mutant ITC would lead to more frequent 2. McClure, W. R. (1985) Annu. Rev. Biochem. 54, 171-204. initiation. The accumulation of CpApU is explained by the 3. Krummel, B. & Chamberlin, M. J. (1989) Biochemistry 28, loss of a fraction of this product before it gets extended with 7829-7842. the next nucleotide. The kinetic barrier for 4. Johnston, D. E. & McClure, W. R. (1976) in RNA Polymerase, CpApUpCpG eds. Losick, R. & Chamberlin, M. (Cold Spring Harbor Lab., extension to CpApUpCpGpA is explained by an exception- Cold Spring Harbor, NY), pp. 101-126. ally high Km of this step even with RNAP"'. The combined 5. Carpousis, A. J. & Gralla, J. D. (1980) Biochemistry 19, 3245- effect of the accelerated oligomer release and a high Km of a 3253. distal step of the abortive reaction explains how the mutation 6. Levin, J. R., Krummel, B. & Chamberlin, M. J. (1987) J. Mol. may inhibit promoter clearance, especially if a high Km step Biol. 196, 85-100. is repeated more than once in the initial transcribed se- 7. Hansen, U. M. & McClure, W. R. (1979) J. Biol. Chem. 254, quence. In accord with this model, RNAP813 displayed di- 5713-5717. minished yield of longer abortive oligomers also in reactions 8. Straney, D. C. & Crothers, D. M. (1987) J. Mol. Biol. 193, primed with ApU (Fig. 2C) as well as with several other 267-278. promoters (data not shown). 9. Metzger, W., Schikor, P. & Heumann, H. (1989) EMBO J. 8, The effect of E813K on elongation can be explained in the 2745-2754. 10. Maizels, M. (1973) Proc. Nati. Acad. Sci. USA 70, 3585-3589. same vein, assuming that the binding site for the incoming 11. Kassavetis, G. A. & Chamberlin, M. J. (1981) J. Biol. Chem. (elongating) substrate is formed, in part, by the nascent 3' 256, 2777-2786. RNA terminus (19). If the mutation affects the holding of the 12. Arndt, K. M. & Chamberlin, M. J. (1990) J. Mol. Biol. 213, 3' terminus, the prevailing consequence for the intrinsically 79-108. stable EC would be an increased Km for elongating rNTP, 13. Lee, D. N., Phung, L., Stewart, J. & Landick, R. (1990)J. Biol. whereas in the unstable ITC the same defect would lead to the Chem. 265, 15145-15153. oligomer loss. This model, if valid, establishes a link between 14. Kashlev, M., Lee, J., Zalenskaya, K., Nikiforov, V. & Gold- the catalytic act of RNA synthesis and the phenomena of farb, A. (1990) Science 248, 1006-1009. abortive initiation and pausing. 15. Zalenskaya, K., Lee, J., Gujuluva, C. N., Shin, Y. K., Slutsky, M. & Goldfarb, A. (1990) Gene 89, 7-12. Besides disrupting the catalytic function, the E813K sub- 16. Lee, J., Zalenskaya, K., Shin, Y. K., McKinney, J. D., Park, stitution somewhat decreases the stability of the RNAP- J. H. & Goldfarb, A. (1989) J. Bacteriol. 171, 3002-3007. promoter complex. The half-life of the RNAP813 and 17. Heumann, H., Metzger, W. & Niehoerster, M. (1986) Eur. J. RNAPWT open complexes determined in the gel-retardation Biochem. 158, 575-579. assay (data not shown) was -10 and 30 min, respectively. 18. Kassavetis, G. A., Zentner, P. G. & Geiduschek, E. P. (1986) This difference is worth noting, even though it cannot ac- J. Biol. Chem. 261, 14256-14265. count for the observed defects in transcription kinetics. It is 19. McClure, W. R., Cech, C. L. & Johnston, D. E. (1978)J. Biol. not inconceivable that the DNA-binding and catalytic func- Chem. 253, 8941-8948. 20. Bourne, H. R., Sanders, D. A. & McCormick, F. (1991) Nature tions partially overlap in the RNAP catalytic center. We (London) 349, 117-127. should also note that both the mutant and the control "wild- 21. Grachev, M. A., Lukhanov, E. A., Mustaev, A. A., Zay- type" RNAP used in our experiments carried the background chikov, E. F., Abdukayumov, M. N., Rabinov, I. V., Richter, Rif-resistant marker RifDJ8, which formally leaves open the V. I., Skoblov, Y. S. & Chistyakov, P. G. (1989) Eur. J. possibility that the Rif mutation contributes to the observed Biochern. 180, 577-585. RNAP813 properties. With this reservation, our observations 22. Jin, D. J. & Gross, C. A. (1991) J. Biol. Chem. in press. Downloaded by guest on September 29, 2021