Identification of a structural element that is essential for two functions of transcription factor NusG

Biochimica et Biophysica Acta 1729 (2005) 135 – 140 http://www.elsevier.com/locate/bba

Identification of a structural element that is essential for two functions of transcription factor NusG Lislott V. Richardson, John P. Richardson* Department of Chemistry, Indiana University, 800 East Kirkwood Avenue, Bloomington, IN 47405, USA Received 1 March 2005; received in revised form 6 April 2005; accepted 8 April 2005 Available online 25 April 2005

Abstract The transcription factor NusG from Escherichia coli modulates the rate of transcript elongation by RNA polymerase and the efficiency of Rho-dependent transcript termination. It consists of two globular domains with an extra loop extending out of the amino-terminal domain in the position that is occupied by a third globular domain in some NusG homologues. We have tested the role of this appended mini-domain by assaying the elongation and termination enhancement activities of variants. The results show that variants with changes in their sequence do not cause a loss of functions, whereas variants with the deletions of the residues in that domain are much less active for both functions. This finding suggests that the mini-domain serves as a structural element for an interaction rather than as a site for residue-specific contacts. D 2005 Elsevier B.V. All rights reserved. Keywords: RNA polymerase; Transcript elongation; Transcript termination; Termination factor Rho

1. Introduction NusG, an essential protein in Escherichia coli and other bacteria, affects the processes of elongation and termination of transcription [1]. It accelerates the rate of RNA chain growth by RNA polymerase [2], is a strong activator of Rho-mediated transcript termination [3,4] and is a component of a complex that causes RNA polymerase to bypass transcript terminators in rrn operons [5,6]. NusG is also known to bind to a single site on Rho factor [7] and to interact with RNA polymerase [8]. However, it apparently does not have a strong affinity for RNA transcripts in the absence of other proteins [9]. Genes encoding proteins that are similar to E. coli NusG have been found in all bacterial and archaeal genomes [10]. In many instances, the nusG gene is part of an operon that encodes SecE homologues [11]. The structure of NusG from Aquifex aeolicus has been determined [12,10]. It and several other NusG proteins contain an insertion of about 70

* Correspondence author. Tel.: +1 812 855 1520; fax: +1 812 855 8300. E-mail address: [email protected] (J.P. Richardson). 0167-4781/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbaexp.2005.04.002

residues that is not present in E. coli NusG. This insertion comprises one of the three distinct domains of the A. aeolicus NusG. The other two domains have sequences that align with sequences in E. coli NusG. Hence, E. coli NusG is proposed to have two main domains, with domain I consisting of the amino terminal residues 1 to 117 and domain II consisting of residues 126 to 182. A flexible segment connects the two domains. The extra domain in A. aeolicus NusG is appended onto domain I between two conserved residues corresponding to Arg-58 and Phe-65 of E. coli NusG on the side of the domain near the point from which the connector segment emerges. The residues in E. coli NusG between Arg-58 and Phe-65 have been modeled as a loop extending out of domain I [12] (Fig. 1); we name it the appended mini-domain. NusG accelerates the rate of RNA polymerase elongation by 20% to 30% over the rate without the factor [2,13]. It does this by stimulating escape of RNA polymerase from Class II pauses [14], which occur at regions where a weak RNA – DNA hybrid allows the RNA polymerase to slide backwards on the template. NusG has little or no effect on Class I pauses, which occur just downstream of sequences that encode a hairpin structure in the nascent RNA, or on the

136

L.V. Richardson, J.P. Richardson / Biochimica et Biophysica Acta 1729 (2005) 135 – 140

role of the structure and sequences in the mini appended domain and of two conserved residues near the amino terminus of domain I.

2. Materials and methods 2.1. Chemicals and reagents

Fig. 1. Domain organization of E. coli NusG. The ribbon diagram is based on the homology model of E. coli NusG, provided by Markus Wahl, and is similar to that shown in Fig. 1B in Reference [12]. It was prepared using the homology model interface of SwissModel available through www.expasy.ch. In this version, the mini-domain is modeled as a 2stranded beta sheet.

rate of polymerization between pause sites [14]. Thus, it could function by reversing backtracking. The effect of NusG on Rho-mediated transcript termination varies considerably depending on the conditions and the specific terminator [15,16,4]. When transcription reactions are carried out under conditions that are optimized for Rho function, NusG has only minimal effects with most terminators [4]. It can enhance the efficiency of termination slightly and cause RNA polymerase to terminate at promoter proximal stop points that are not normally used with Rho alone. However, in cells that have been depleted of NusG, the efficiency of termination at several Rhodependent terminators is much lower than in the cells with normal levels of NusG [3]. This result implies that NusG serves as a very important factor in vivo. NusG also significantly enhances termination at some sites in a reaction mixture in which the concentration of the 4 NTPs mimics the concentration in the cell [4]. Under those conditions, transcript elongation is relatively fast and Rho is not very effective by itself. Indeed, one function that NusG has on the process of transcript termination is to increase the rate with which Rho dissociates transcripts from a ternary transcription complex [17]. The fact that NusG can bind to both RNA polymerase and to Rho could allow it to serve as a bridge to assist the formation of a productive complex between Rho and its entry site on a nascent transcript. On the other hand, because NusG affects the dynamics of pausing at some pause sites and since the stop points in a Rho-dependent terminator are at pause sites, proposals have been made that relate the effects of NusG on pausing with its effects on Rho function [7,13]. To learn more about how the structure of NusG affects its functions in transcript elongation and Rho-mediated termination, we have prepared mutant forms of NusG and tested the effects of these variant proteins on the overall rate of transcript elongation and the modulation of Rhodependent termination in vitro. Specifically, we test the

DNA oligonucleotides were ordered from Integrated DNA Technology, Inc., Coralville, IA. pET11a was purchased from Novagen. Nucleoside triphosphates and deoxynucleoside triphosphates were from Amersham; 32Plabelled nucleotides were from ICN, and ApU was from Sigma. All other chemicals were reagent grade. 2.2. Construction of plasmids encoding NusG pET11a/NusG, a 6212 bp plasmid, was constructed by the insertion of a nusG-gene DNA fragment between the NdeI and BamHI sites of pET11a. The sequence was amplified from E. coli DNA by PCR using the 5V primer (5V GACTcatatgTCTGAAGCTCCTAAAAAGC 3V) encoding the NdeI site (in italics) followed by a sequence segment from nusG and the 3V primer (5V GACTggatccATCGCTGGGTTAGGC 3V) encoding the BamHI site (in italics), followed by a sequence segment ending 25 nucleotides downstream from the nusG stop codon. After the digestion of both the vector and the fragment with NdeI and BamHI, the vector was further treated with calf intestine phosphatase. Both samples were extracted with phenol and chloroform/isoamyl-alcohol and purified by gel electrophoretic separation. After treatment with ligase, the resulting plasmid was introduced into E. coli JM107, and the sequence of the inserted nusG confirmed by DNA sequencing. Mutant forms of this plasmid encoding the NusG variants, W9L, Y10A, R57A, R58AD59 –61 (deleting the sequence encoding KSE), Ala-rich mini (with residues 57 to 61 (RRKSE) changed to ARAAA), and D52 –61 (deleting the sequence encoding IRGGQRRKSE), were prepared by the Stratagene QuikChange\ procedure. The sequences of all constructs used in this study were confirmed by DNA sequencing. 2.3. Expression and isolation of NusG One half liter of Maximum Induction Medium [18] with 100 Ag/ml ampicillin was inoculated with a 5 ml overnight culture and cultivated at 37 -C in a rotary shaker. After reaching a cell density of 2  108 cells/ml, mGP1-2 phage [19] was added at a multiplicity of about 10 and shaking was continued at a reduced speed (100 rpm) to let the phage adhere to the cells. This phage contains a gene encoding the T7 RNA polymerase under the control of the E. coli lac promoter. Isopropyl-h-d-thiogalactoside was added 15 min later at a concentration of 1 mM. The cells were harvested 3

L.V. Richardson, J.P. Richardson / Biochimica et Biophysica Acta 1729 (2005) 135 – 140

h later by centrifugation at 4800  g for 10 min, washed with 50 mM Tris – HCl (pH 7.6) and stored frozen at 80 -C. The yield of wet, packed cells was about 3 g. To monitor the expression of the NusG protein, small samples were taken at 1 h intervals, centrifuged, washed with 5 mM Tris – HCl, pH 6.8, resuspended in the same buffer and boiled for 5 min after the addition of 4  loading buffer and then analyzed by SDS-PAGE with a 12.5% gel. NusG was purified as described by Passman and von Hippel [7] with the following alterations: the ammonium sulfate pellet (from about 3 g of cells) was re-suspended in 4.0 ml buffer Q (10 mM Tris –HCl, pH 7.8, 1 mM EDTA, 1 mM dithiothreitol, and 5% glycerol) and dialyzed overnight against four changes of 250 ml buffer Q. The sample was centrifuged at 4000  g for 10 min. It was then applied to a Toyopearl\ DEAE column (2.5  12 cm), equilibrated with buffer Q. After extensive washing, NusG was eluted with a 100 ml 0 – 0.5 M NaCl linear gradient in buffer Q. The peak fractions were analyzed on a 12.5% SDS PAGE. The purest fractions were pooled and dialyzed overnight against buffer Q with 50% (v/v) glycerol and stored at 20 -C. All NusG preparations were greater than 95% pure. The concentrations of the purified NusG were determined by the using the Bradford assay. Some of the mutant proteins (W9L, Y10A and D52 – 61) were purified from inclusion bodies. In these cases, the pellet that resulted from centrifugation of the sonicated cells was resuspended in 0.5 ml buffer Q containing 6 M guanidine hydrochloride and the solution centrifuged for 30 min at 10,000  g. The supernatant was saved and the pellet re-extracted with 0.5 ml of the same solution. The centrifugation was repeated and the two supernatant fractions were combined. Small samples from the supernatant and pellet fractions were analyzed by SDS-PAGE. The combined supernatant fraction was gradually diluted by the addition with constant mixing at 0 -C of 1, 2, 4, 8, 16 and 32 ml of buffer Q, respectively, with 5 to 10 min intervals between the successive additions. The final volume was 64 ml. After a 10-min centrifugation at 4000  g, ammonium sulfate was slowly added to a final level of 50% saturation and the slurry stirred in the cold for 30 min. After centrifugation at 4000  g for 30 min, the pellet was resuspended in 500 Al of buffer Q and the sample was filtered on a 0.5 cm  35 cm Bio-Gel 0.5 column in buffer Q. The peak fractions were analyzed by SDS-PAGE. Fractions with greater than 95% purity were pooled and then dialyzed overnight against buffer Q with 50% (v/v) glycerol. The concentration of the final product was determined using the Bradford assay. 2.4. Transcription assays Hexahistidine-tagged E. coli RNA polymerase was purified from strain RL916 kindly provided by Robert Landick (University of Wisconsin) by the procedure of Kashlev et al. [20] with modifications provided by Robert

137

Landick. E. coli Rho factor was purified as described in [18]. Termination assays were performed as described in [21] except that [a-32P]CTP (5 ACi/nmol, 25 AM) was used instead of [a-32P]UTP. The DNA template carrying the entire bacteriophage E cro gene was prepared by PCR amplification from the plasmid pKKCYC [21]. Rates of transcript elongation were measured using a DNA template containing the ops pause site from E. coli pheP placed between the T7A1 promoter and the E. coli his attenuator terminator [14]. The template was prepared by PCR amplification from plasmid pIA251 [14] kindly provided by Irina Artsimovitch (Ohio State University). [a-32P]CMPlabeled transcription complexes halted at position A29 by UTP deprivation [14] were mixed at 37 -C with a solution to bring GTP to 15 AM, ATP, CTP, and UTP to 150 AM each, heparin to 0.1 mg/ml, and NusG (as indicated) to 50 nM. Samples were removed at various times, mixed with a stop solution [21] and separated by gel electrophoresis. Rates of RNA chain growth were determined by dividing the time required for half of the elongating transcripts to be extended from the arrest site at base-pair 29 to the termination site at base-pair 138 into the number of RNA nucleotides added (109). The relative amounts of the radioactive transcripts was measured by quantitation with ImageQuant software (Amersham Biosciences) of PhosphoImager plates exposed to the polyacrylamide gels used to separate the transcripts.

3. Results In E. coli NusG, the sequence of the appended minidomain contains several basic residues. Although the sequence of residues at the region of the mini-domain is not conserved, the relatively high frequency of basic residues is a common feature of NusG orthologs in that region. We thus prepared some mutant forms of E. coli NusG with changes in that region. These included a change of a single arginine residue to alanine (R57A), a deletion of some of the region (R58AD59 – 61), a change of the sequence of residues 57 to 61 (RRKSE) to ARAAA (Alarich mini) and a deletion of the whole mini-domain (D52– 61). As well, we tested two mutants with changes of the conserved tryptophan and tyrosine residues at positions 9 and 10, respectively (W9L and Y10A). All the proteins could be expressed in high yields in a soluble form, except D52– 61 and Y10A. They were prepared by re-folding the protein extracted from inclusion bodies. To test the effects of the variant NusG on transcript elongation, we used a model transcription system containing a known Class II pause site, the ops pause of the E. coli pheP gene [14], placed between the T7 A1 promoter and an intrinsic terminator (from the E. coli his attenuator). The rate of elongation was determined by measuring the time for half of the RNA polymerase molecules starting from an arrest site at position 29 to reach the terminator upon the addition of all four NTPs. Fig. 2 shows the size distributions of

138

L.V. Richardson, J.P. Richardson / Biochimica et Biophysica Acta 1729 (2005) 135 – 140

Fig. 2. Effects of NusG and a mutant NusG on RNA chain elongation through an ops pause site. 32P-labeled A29 complexes on a template with the PheP ops pause site (at 62 nt) were incubated at 37 -C with 15 AM GTP, 150 AM each of ATP, CTP, and UTP in the presence of the indicated NusG (50 nM). Samples were taken at the times (in s) indicated above the lanes. The lengths of the indicated transcripts are 62 nucleotides for P (pause transcript), 138 nucleotides for T (terminated transcript) and 369 nucleotides for RO (run-off transcript).

transcripts as a function of time after the addition of the NTPs to the reaction mixtures without NusG, with wild-type NusG and with a representative mutant. The effect of NusG on elongation is evident from the fact that the times for nearly equivalent numbers of polymerase molecules to reach the terminator were 40 s and 30 s in the absence and presence of wild-type NusG, respectively. The rates, as determined from quantitative analyses of these and other transcription results, are presented in Table 1. In this system, NusG enhances the rate of elongation by about 1.6 fold. In contrast, the representative mutant shown in Fig. 2, R58AD59– 61, which has a partial deletion of the minidomain, enhanced the elongation rate to a much lower extent. A very similar result was found with the mutant lacking all of the mini-domain (D52 –61) (Table 1). In contrast, a mutant with a change of one of the basic residues in the region to an uncharged residue (R57A) or a mutant with a more radical change in the sequence of the residues in the region (Ala-rich mini) was fully active in enhancing elongation. These results suggest that the presence of an Table 1 Effects of NusG factors on rates of RNA chain growth NusG

Rate (nucleotides/s)

None Wild type R57A Ala-rich mini. D52 – 61 R58AD59 – 61 W9L Y10A

2.6 T 0.2 4.1 T 0.2 4.5 T 0.3 4.5 T 0.3 2.7 T 0.2 2.9 T 0.3 4.5 T 0.3 2.9 T 0.3

altered sequence in the appended mini-domain does not affect this activity of NusG, whereas the deletion of all or part of the sequence is quite deleterious. In the tests of the two highly conserved residues near the N terminus of NusG, the change of tryptophan at position 9 to a leucine had no effect on the rate enhancement function, whereas the change of the tyrosine-10 to alanine was quite deleterious (Table 1). Because two of the mutants lacking the transcript elongation enhancement activity, D52– 61 and Y10A, had to be purified after re-folding of the protein extracted from inclusion bodies with guanidine hydrochloride, we examined the CD spectra from 195 to 300 nm of NusG and the mutant variants used in this study. We found that the spectra of the mutants, including those isolated from inclusion bodies, were identical to that of the wild type (data not shown). Hence, we conclude that most, if not all, of the secondary structural elements of the mutants have formed normally. Also, after re-folding, the proteins isolated from the inclusion bodies remained soluble and stable during purification and storage. In the case of W9L, the re-folding procedure yielded an active protein. These results suggest that the lack of activity with D52– 61 and Y10A was not because of an inability for the two main domains of the protein to fold normally in those proteins. From the result with Y10A, we conclude that the ability of NusG to enhance transcription elongation also depends on the specific structure at the N-terminus of domain I. To test the effects of the changes on the ability of the NusG variants to enhance Rho factor function, we measured the relative yields of terminated RNA molecules produced during the transcription of the E cro gene DNA sequence on

L.V. Richardson, J.P. Richardson / Biochimica et Biophysica Acta 1729 (2005) 135 – 140

a fragment that ended just beyond subsite III of tR1, a prototypical Rho-dependent terminator [22,23]. Under the conditions used, Rho in the absence of NusG caused approximately 60% of the RNA polymerase molecules to terminate transcription at subsites I, II and III of tR1, with 38% terminating at subsite I or in the tRE region [24] (Fig. 3). When wild-type NusG was also present in the reaction mixture, the fraction of RNA polymerases terminating at subsite I and in tRE was 55%. This enhancement of termination at the earlier sites is characteristic of the effect of NusG in this system. Again, three of the mutants, R57A, Ala-rich mini and W9L, were just as active as wild-type NusG in enhancing termination, whereas the two mutants with deletions of the mini-domain were partially defective in this activity. The Y10A mutant was also totally inactive in this assay. With this set of mutants, there is a perfect correlation between the effects of the changes on the two assays. All the mutants that are defective in enhancing elongation are also defective in enhancing termination, whereas all the mutants that are effective in enhancing elongation are effective in enhancing termination.

4. Discussion We have found that a part of E. coli NusG that has been ˚ out of modeled as a loop that extends by as much as 28 A the globular domain I is important for its functions in transcription elongation and termination (Fig. 1). This importance is suggested by the finding that variants of NusG with deletions of some or all of the residues of this

Fig. 3. Effects of NusG and several mutant forms of NusG on termination at k tR1. 32P-labeled transcription products of E cro DNA were prepared in the presence of 20 nM Rho with 50 nM of the indicated NusG proteins. The reaction mixture for the first lane on the left had no Rho or NusG. The indicated transcripts are: RT, read-through to end of template (378 nucleotides); tR1 I, terminated at subsite I (295 nucleotides); and tRE, terminated in the region preceding tR1 (245 to 280 nucleotides). The numbers under each line are the percent of transcripts terminated in tRE and at tR1 subsite I. The errors for those values are T 4%.

139

appended mini-domain were very deficient in functions. However, because a variant in which the sequence of the mini-domain had been changed extensively had normal functions, we conclude that the mini-domain serves a role as a structural element, such as a post or a stalk, rather than as a site for residue-specific contacts. This view is consistent with a phylogenetic comparison of the sequences of the corresponding mini-domain residues in the NusG proteins from other bacteria [12]. In the NusG proteins without a full extra domain, the number of residues between the elements of the globular part of domain I upon which the minidomain is appended is the same, but the sequences of those residues vary considerably. The full extra domain found in A. aeolicus NusG (called domain II for that protein) is a beta barrel that extends from domain I about as far as an appended mini-domain would extend if it were a beta hairpin structure. Hence, the extra domain could be just as a more substantial ‘‘stalk’’ than is the mini-domain of E. coli NusG. Deletions of residues in the mini-domain could also be causing loss of activity because a change in its structure affects in some subtle way the structure of domain I. The finding that the mutation of tyrosine-10 to alanine has the same effect on the functions of NusG as the mutations that deleted part of the mini-domain suggests that the structure of domain I itself is important for NusG function in transcription and termination. Tyr10 is on the beta strand that is the first secondary structural element of domain I [10,12] and is situated on the opposite side of the domain from the mini-domain (Fig. 1). Steiner et al. [12] have noted that domain I is a structural homologue of the 30S ribosomal protein S6, a protein that contacts both RNA and another ribosomal protein, S18. Hence, subtle changes in the structure of domain I could be affecting the interactions of NusG with another protein, such as RNA polymerase, or with RNA. The finding that all the mutants with defects were nearly equally deleterious for the two functions suggests that their changes affect an interaction that is needed for both. NusG is known to bind to both Rho and RNA polymerase [7,8,15], and one mechanism that has been suggested for how NusG can act to enhance Rho function is to serve as a bridge to assist in its interactions with the nascent RNA emerging from RNA polymerase [15,25]. With this model, a mutation that would affect the binding of NusG to RNA polymerase would be deleterious both for its effects on transcriptional elongation and Rho-mediated termination. Another possibility is that the effect of NusG on Rho-dependent termination could be mechanistically tied to its effect on transcriptional elongation. For instance, Passman and von Hippel [7] have suggested a way in which NusG could enhance both the rate of elongation and Rho-mediated termination is by preventing backsliding by RNA polymerase. Because none of the mutants analyzed in this study affected one without the other, that hypothesis still remains perfectly valid.

140

L.V. Richardson, J.P. Richardson / Biochimica et Biophysica Acta 1729 (2005) 135 – 140

Acknowledgements We thank Markus Wahl for providing us with the pdb file of the homology-model of E. coli NusG, Robert Landick for E. coli RL916, and Irina Artsimovitch for pIA251. This research was supported by grant GM 56095 from the NIGMS, U.S. Department of Public Health and Human Services.

[11]

[12]

[13]

[14]

References [1] J.P. Richardson, J. Greenblatt, Control of RNA chain elongation and termination, in: F.C. Neidhardt, et al., (Eds.), Escherichia coli and Salmonella: Cellular and Molecular Biology, ASM Press, Washington, 1966, pp. 822 – 848. [2] E. Burova, S.C. Hung, V. Sagitov, B.L. Stitt, M.E. Gottesman, Escherichia coli NusG protein stimulates transcription elongation rates in vivo and in vitro, J. Bacteriol. 177 (1995) 1388 – 1392. [3] S.L. Sullivan, M.E. Gottesman, Requirement for E. coli NusG protein in factor-dependent transcription termination, Cell 68 (1992) 989 – 994. [4] C.M. Burns, J.P. Richardson, NusG is required to overcome a kinetic limitation to Rho function at an intragenic terminator, Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 4738 – 4742. [5] C.L. Squires, J. Greenblatt, J. Li, C. Condon, C.L. Squires, Ribosomal RNA antitermination in vitro: requirement for Nus factors and one or more unidentified cellular components, Proc. Natl. Acad. Sci. U. S. A. 90 (1993) 970 – 974. [6] M. Torres, J.-M. Balada, M. Zellars, C. Squires, C.L. Squires, In vivo effect of NusB and NusG on rRNA transcription antitermination, J. Bacteriol. 186 (2004) 1304 – 1310. [7] Z. Passman, P.H. von Hippel, Regulation of Rho-dependent transcription termination by NusG is specific to the Escherichia coli elongation complex, Biochemistry 39 (2000) 5573 – 5585. [8] J. Li, R. Horwitz, S. McCracken, J. Greenblatt, NusG, a new Escherichia coli elongation factor involved in transcriptional antitermination by the N protein of phage E, J. Biol. Chem. 267 (1992) 6012 – 6019. [9] J.R. Nodwell, J. Greenblatt, Recognition of boxA antiterminator RNA by the E. coli antitermination factors NusB and ribosomal protein S10, Cell 72 (1993) 261 – 268. [10] J.R. Knowlton, M. Bubunenko, M. Andrykovitch, W. Guo, K.M. Routzahn, D.S. Waugh, D.L. Court, X. Ji, A spring-loaded state of NusG in its functional cycle is suggested by X-ray crystallography

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22] [23]

[24]

[25]

and supported by site-directed mutants, Biochemistry 42 (2003) 2275 – 2281. C.J. Ingham, P.A. Furneaux, Mutations in the h subunit of the Bacillus subtilis RNA polymerase that confer both rifampicin resistance and hypersensitivity to NusG, Microbiology 146 (2000) 3041 – 3049. T. Steiner, J.T. Kaiser, S. Marinkovic, R. Huber, M.C. Wahl, Crystal structure of transcription factor NusG in light of its nucleic acid- and protein-binding activities, EMBO J. 21 (2002) 4641 – 4653. C.M. Burns, L.V. Richardson, J.P. Richardson, Combinatorial effects of NusA and NusG on transcription elongation and Rho-dependent termination in Escherichia coli, J. Mol. Biol. 275 (1998) 307 – 316. I. Artsimovitch, R. Landick, Pausing by bacterial RNA polymerase is mediated by mechanistically distinct classes of signals, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 7090 – 7095. J. Li, S.W. Mason, J. Greenblatt, Elongation factor NusG interacts with termination factor q to regulate termination and antitermination of transcription, Genes Dev. 7 (1993) 161 – 172. K.W. Nehrke, F. Zalatan, T. Platt, NusG alters rho-dependent termination of transcription in vitro independent of kinetic coupling, Gene Expr. 3 (1993) 119 – 133. C.M. Burns, W.L. Nowatzke, J.P. Richardson, Activation of Rhodependent transcription termination by NusG, J. Biol. Chem. 274 (1999) 5245 – 5251. W. Nowatzke, L. Richardson, J.P. Richardson, Purification of transcription termination factor Rho from Escherichia coli and Micrococcus luteus, Methods Enzymol. 274 (1996) 353 – 363. S. Tabor, Expression using the T7 RNA polymerase/promoter system, in: F.M. Ausubel, et al. (Eds.), Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, New York, NY, 1991, pp. 16.2.1 – 16.2.8. M. Kashlev, E. Martin, A. Polyakov, K. Severinov, V. Nikiforov, A. Goldfarb, Histidine-tagged RNA polymerase: dissection of the transcription cycle using immobilized enzyme, Gene 130 (1993) 9 – 14. L.V. Richardson, J.P. Richardson, Rho-dependent termination of transcription is governed primarily by the upstream rho utilization (rut) sequences of a terminator, J. Biol. Chem. 271 (1996) 21597 – 21603. L.F. Lau, J.W. Roberts, R. Wu, Transcription terminates at EtR1 in three clusters, Proc. Natl. Acad. Sci. U. S. A. 79 (1982) 6171 – 6175. W.D. Morgan, D.G. Bear, P.H. von Hippel, Rho-dependent termination of transcription I: identification and characterization of termination sites for transcription from the bacteriophage k PR promoter, J. Biol. Chem. 258 (1983) 9553 – 9564. R.S. Washburn, S.L. Stitt, In vitro characterization of transcription termination factor Rho from Escherichia coli rho(nusD) mutants, J. Mol. Biol. 260 (1996) 332 – 346. K.W. Nehrke, T. Platt, A quaternary transcription termination complex, J. Mol. Biol. 243 (1994) 830 – 839.