Cold Shock Induces a Major Ribosomal-Associated Protein That Unwinds Double-Stranded RNA in Escherichia Coli PAMELA G

Proc. Natl. Acad. Sci. USA Vol. 93, pp. 76-80, January 1996 Biochemistry Cold shock induces a major ribosomal-associated protein that unwinds double-stranded RNA in Escherichia coli PAMELA G. JONES, MASANORI MITTA, YOUNGHO KIM, WEINING JIANG, AND MASAYORI INOUYE Department of Biochemistry, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ 08854 Communicated by Carol A. Gross, University of California, San Francisco, CA, September 20, 1995

ABSTRACT A 70-kDa protein was specifically induced in expression of certain genes including heat-shock proteins only Escherichia coli when the culture temperature was shifted at low temperature. We propose that at low temperature this from 37 to 15°C. The protein was identified to be the product DEAD-box protein with helix-destabilizing activity is essential of the deaD gene (reassigned csdA) encoding a DEAD-box for ribosomal function to increase translational efficiencies of protein. Furthermore, after the shift from 37 to 15°C, CsdA mRNAs by unwinding stable secondary structures formed at was exclusively localized in the ribosomal fraction and became low temperature. a major ribosomal-associated protein in cells grown at 15°C. The csdA deletion significantly impaired cell growth and the MATERIALS AND METHODS synthesis of a number of proteins, specifically the derepression of heat-shock proteins, at low temperature. Purified CsdA Bacterial Strains and Media. E. coli strains SB221 (7), was found to unwind double-stranded RNA in the absence of JC7623 (recB recC sbcB) (13), and the corresponding csdA ATP. Therefore, the requirement for CsdA in derepression of mutant were used in this study. heat-shock protein synthesis is a cold shock-induced function Cultures for pulse labeling were grown in M9 medium possibly mediated by destabilization of secondary structures supplemented with 19 amino acids (no methionine) and 4 previously identified in the rpoH mRNA. bases as described (14). Cultures used for the isolation of ribosomes were grown in LB medium. Bacterial adaptation to various environmental stresses has Radioactive Labeling of Cultures. Steady-state cultures of been extensively investigated (reviewed in refs. 1-4). Interest- bacterial strains were grown at 37°C to an optical density at 420 ingly, it has been demonstrated that Escherichia coli has an nm to -0.50 and the culture temperature was then shifted to not to a 15°C. A 1-ml portion of the culture was labeled with trans- adaptive response only high temperature by inducing [35S]methionine (1096 Ci/mmol; 60 mCi/ml; I Ci = 37 GBq; group of heat-shock proteins but also to low temperature by Amersham) at the times indicated. Extracts were prepared and inducing a group of cold-shock proteins (5, 6). In contrast to processed by two-dimensional polyacrylamide gel electro- heat-shock proteins, which include protein chaperones re- phoresis as described (15). quired for protein folding and peptidases, cold-shock proteins Preparation of 70S Ribosomes. Ribosomes were isolated as appear to be involved in various cellular functions such as described (16) from a 100-ml culture of E. coli SB221 cells transcription, translation, and DNA recombination (5, 6). grown at 37°C, 3 hr after a shift from 37 to 15°C, or 27 hr after Among the cold-shock proteins of E. coli, CspA has been a shift from 37 to 15°C. Proteins were extracted according to identified as the major cold-shock protein, which is almost the acetic acid method (17) and were precipitated with ace- exclusively produced at low temperature at a level of 250,000 tone. CsdA was dissociated from the 70S ribosome in the molecules per cell (5, 7). The three-dimensional structure of presence of 1 M NH4Cl and precipitated by dialysis against 60 CspA consisting of 69 amino acid residues has been deter- mM NH4Cl. CsdA was dissolved in buffer containing 1 M mined, which is composed of five antiparallel (3-sheet struc- NH4Cl and was passed through a phenyl-Sepaharose column tures (8, 9). CspA binds to single-stranded DNA (8), and its equilibrated in the same buffer. Purified CsdA was eluted with possible function as an RNA chaperone has been speculated buffer containing 0.1 M NH4Cl. (6). In addition to CspA, E. coli contains a large family of Helicase Assay. The standard double-stranded RNA CspA-like proteins consisting of CspB, CspC, CspD, and CspE, (dsRNA) substrate used for the assay was prepared as de- among which only CspB is a cold-shock protein (10, 11). scribed (18). This substrate consists of a 98-base RNA hybrid- In the present paper, we report a newly discovered cold- ized to a radiolabeled 38-base RNA at a 29-base central shock protein of 70 kDa, which is also almost exclusively complementary region forming a dsRNA. Together with the produced upon a temperature shift from 37 to 15°C, similar to substrate and purified CsdA, the RNA helicase reaction the induction of CspA. It was found that this newly identified mixture contained 20 mM Hepes-KOH (pH 7.6), 2 mM cold-shock protein is exclusively localized in the ribosomal dithiothreitol, 150 mM KCl, 1 mM MgCl2 in the absence or fraction and became a major ribosomal-associated protein at presence of 1 mM ATP. The mixture was incubated at 37°C for low temperature. This protein was purified and identified to be 30 min followed by the addition of 5 Al of a stop solution the product of the gene that has been known as deaD. This containing 0.1 M Tris HCl (pH 7.4), 20 mM EDTA, 0.5% SDS, gene had been isolated as a multicopy suppressor for a 0.1% Nonidet P-40, 0.1% bromophenol blue, 0.1% xylene temperature-sensitive mutation located in the gene encoding cyanol, and 50% (wt/vol) glycerol. Aliquots of each reaction ribosomal S2 protein and proposed to encode a putative mixture were subjected to SDS/polyacrylamide gel electro- ATP-dependent RNA helicase based on sequence similarities phoresis. with other known DEAD-box proteins (12). This protein now is assigned CsdA for cold-shock DEAD-box protein A. We found that this protein has a helix-destabilizing activity. Dis- RESULTS ruption of the gene resulted in a defect in growth and in Identification of a 70-kDa Cold-Shock Protein. Previously, cold-shock proteins were identified by two-dimensional gel The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in Abbreviations: dsRNA, double-stranded RNA; ORF, open reading accordance with 18 U.S.C. §1734 solely to indicate this fact. frame. 76 Downloaded by guest on September 26, 2021 Biochemistry: Jones et al. Proc. Natl. Acad. Sci. USA 93 (1996) 77

A 370C B 370C--150C A Major Ribosomal-Associated Protein at Low Tempera- ture. Using the cell extract labeled with [35S]methionine at 15°C as described above, we found that the 70-kDa protein is

...... localized in the supernatant fraction after centrifugation at 28,000 x g for 45 min (data not shown). When this cytoplasmic ...... '.... : fraction was further fractionated by ultracentrifugation at # :'''..,..''.:.,'.4e 4-WS. 138,000 x g for 3 hr to isolate the ribosomal fraction, the cold-shock protein was almost quantitatively recovered in the ribosomal fraction. The analysis of the ribosomal proteins by two-dimensional gel electrophoresis is shown in Fig. 2. The 70-kDa cold-shock protein was not detected in the ribosomal fraction from the culture at 37°C (Fig. 2A), while it was found in 70S ribosomes from the culture at 15°C (Fig. 2B). Further- more, the cold-shock protein became a major component of 70S ribosomes during steady-state growth at 15°C (Fig. 2C). FIG. 1. Total cellular protein analysis before and after cold-shock When 70S ribosomes were fractionated into 30S and 50S treatment. Cells were labeled with trans-[35S]methionine as described. subunits, CsdA was found to be associated with both subunits Total cellular proteins were analyzed by two-dimensional gel electro- (data not shown). When 1 M NH4Cl was added to the phoresis. The first dimension was carried out between the pH range of cold-shock ribosomal fraction, 80-90% of the 70-kDa protein 3.5 (right) and 10 (left). (A) Cells were labeled for 5 min at 37°C. (B) was dissociated from ribosomes (data not shown), suggesting Cells were labeled between 30 and 45 min after temperature shift to that this protein is associated with the ribosomes by ionic 15°C. CsdA is indicated by the small arrows, CspA is boxed, and ribosomal proteins are indicated by the large arrows. interactions. The present results indicate that CsdA is an auxiliary ribosomal protein, which is probably associated with electrophoresis in the pH range 4-7 (5). However, when the both 30S and 50S ribosome subunits. On the basis of relative sample labeled with [35S]methionine for 30 min after the shift intensity of the CsdA spot to spots of other ribosomal proteins, from 37 to 15°C was analyzed by two-dimensional gel electro- it seems that the proteins are present in approximately stoi- phoresis in the pH range 3-10, a major protein of 70 kDa was chiometric amounts. detected at basic pH as shown by an arrow in Fig. 1B. This CsdA Is a Helix-Destabilizing Protein. CsdA has been protein is not detectable in the control cells incubated at 37°C proposed to be a putative ATP-dependent helicase, because it (Fig. 1A). The pl value of the 70-kDa cold-shock protein was contains several highly conserved motifs including the DEAD estimated to be -9. Note that under the 3-10 pH range used, motif (12). To determine whether CsdA is indeed an ATP- the synthesis of a number of ribosomal proteins (indicated by dependent helicase, CsdA was purified to homogeneity from large open arrows in Fig. 1) also increased. the ribosomal fraction as described and tested for RNA The protein was extracted from the gel and subjected to helicase activity using the standard dsRNA substrate (18). This sequential analysis by Edman degradation. The sequence was substrate consists of 98-base and 38-base RNAs. The 29-base determined to be AEFETTFADLGLKAPILEAL, which was 3'-terminal sequence of the 38-base RNA hybridizes to a found to be identical to the internal sequence starting from the central 29-base sequence of the 98-base RNA to form a alanine residue at position 18 of the open reading frame dsRNA. Only the 38-base RNA was labeled with 32p. In Fig. (ORF) of the gene previously designated deaD (12). This gene 3, the dsRNA substrate migrated in position in a 17% poly- has been identified as a multicopy suppressor of a tempera- acrylamide gel (lanes 1 and 3). When the substrate was ture-sensitive mutant of ribosomal S2 protein (12). The au- incubated at 95°C followed by quick cooling, the unannealed thors found that the ORF encoded by the DNA fragment 38-base RNA labeled with 32p migrated to position b (lane 2). contains several motifs, including the DEAD motif, highly The addition of increasing concentrations of CsdA to the conserved in RNA and DNA helicases (reviewed in ref. 19). assay mixture containing dsRNA and ATP (1 mM) resulted in Accordingly, they speculated that the ORF encodes an ATP- increasing unwinding of dsRNA substrate as demonstrated by dependent RNA helicase. This protein is now called CsdA the appearance of band b (Fig. 3, lanes 4-8). To determine the (cold-shock DEAD-box protein A). Since there is a methio- ATP requirement, the helicase assay was performed using the nine residue at position 17 in the ORF immediately upstream same conditions as in lane 8 except without ATP. CsdA was of the N-terminal alanine residue of the 70-kDa cold-shock still able to cause the unwinding of dsRNA in the absence of protein, this methionine residue is now reassigned as the ATP (lane 9), indicating that CsdA has helix-destabilizing initiation codon for the protein. activity rather than helicase activity.

A 370C B 150C, 3 hr C 15°C, steady state

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FIG. 2. Two-dimensional analysis of 70S ribosomal proteins isolated from 37°C and cold-shocked cells. Ribosomes were prepared as described. Two-dimensional gel electrophoresis was carried out under the same conditions as in Fig. 1. Gels were stained with Coomassie blue. (A) 70S ribosomes from cells exponentially grown at 37°C. (B) 70S ribosomes from cells incubated for 3 hr after a shift from 37 to 15°C. (C) 70S ribosomes from cells exponentially grown for 27 hr after a shift from 37 to 15°C. CsdA is indicated by arrows. Downloaded by guest on September 26, 2021 78 Biochemistry: Jones et al. Proc. Natl. Acad. Sci. USA 93 (1996) 1 2 3 4 5 6 7 8 9 temperature and that its function is required for normal cell division at low temperature. CsdA Requirement for Heat-Shock Gene Expression at Low Temperature. To examine the physiological role of csdA on global gene expression, the parent and csdA mutant were at and a _ labeled with [35S]methionine 37°C and 15°C protein patterns were analyzed by two-dimensional gel electrophoresis. With the notable exception of the synthesis of CsdA, no discernible differences were observed between the mutant and the parent strain during exponential growth at 37°C (data not shown) and also during an early period of the cold-shock box treatment between 30 and 45 min after the shift to 15°C (see Figs. 4 and 6A and B). However, when cells were labeled 3 hr after the shift, differences in gene expression became evident as shown in Fig. 6 C and D. Note that after 3 hr of incubation at 15°C, cells had recovered from cold-shock stress and were FIG. 3. RNA unwinding activity of CsdA. The helicase reaction was some performed in the presence of purified CsdA in the presence (lanes in exponential growth (data not shown). Although 4-8) or absence (lane 9) of 1 mM ATP. Lanes 1 and 3, substrate proteins were made at similar rates of synthesis in the mutant dsRNA; lane 2, unannealed single-strand RNA as a result of incuba- (Fig. 6D) and parent strain (Fig. 6C), some other proteins were tion at 95°C and quick cooling of the dsRNA substrate. Lanes 4-9, with made at much lower rates including major heat-shock proteins 10, 20, 50, 100, 200, and 200 ng of CsdA protein, respectively. DnaK and GroEL (enclosed in upper and lower circles,

CsdA Requirement for Optimal Growth at Low Tempera- A ture. The fact that CsdA is a helix-destabilizing protein and associates with the ribosome at low temperature indicates a specialized role for CsdA in translation at low temperature. To better understand the physiological role of CsdA at low temperature, a csdA deletion mutant was constructed by linear DNA transformation using a recB recC sbcB strain (13). The deletion ofcsdA was confirmed by Southern blot analysis of the chromosomal DNA (data not shown). As shown in Fig. 4, the induction of CsdA synthesis became undetectable in the csdA mutant in contrast to the parent csdA + strain upon a temperature shift from 37 to 150C. At 370C, no major differences in growth rate were observed between the csdA- mutant and its parent (data not shown). In contrast, in exponential growth at 15°C, the growth of the mutant cells was significantly impaired, B for the mutant grew 2 times slower than the parent cells. The doubling time for the mutant was 16 hr in contrast to 8 hr for the parent strain (data not shown). Furthermore, after 96 hr at 150C, the mutant formed long filamentous cells (Fig. 5 B and C), while the parent strain did not (Fig. 5A). These results indicate that CsdA plays a key role in optimal cell growth at low

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sri. anI)cd FIG. 4. Two-dimensional gel electrophoresis of total cellular proteins from csdA + and csdA cells following a shift from 37 to 15°C. The csdA gene was disrupted by linear DNA transformation using strain JC 7623 (recB recC sbcB). Cells were labeled with trans-[35S]methionine for 15 min at 30 min after the temperature shift from 37 to 15°C. Total cell extracts were analyzed by two-dimensional gel electrophoresis under the same conditions as in Fig. 1. Autoradiograms of the two-dimensional gel for csdA + cells (A) and for csdA cells (B). CsdA is indicated by arrows. Downloaded by guest on September 26, 2021 Biochemistry: Jones et al. Proc. Natl. Acad. Sci. USA 93 (1996) 79 A B shown in Fig. 6, the addition of 4% ethanol resulted in a significantly higher level of heat-shock protein synthesis in the csdA+ strain (compare Fig. 6A with 6E) compared to the csdA strain (compare Fig. 6B with 6F), -4-fold higher expression in both DnaK and GroEL as measured by densitometry. There- 0. fore, CsdA is required for normal derepression of heat-shock protein synthesis, even in the presence of ethanol at low Ai *i temperature. The impairment of heat-shock gene expression ..l-c of the csdA4 strain was only at low temperature, since heat induction of dnaK and groEL observed by shifting the culture from 28 to 42°C occurred in the csdA strain at a comparable level to that of the csdA+ strain (data not shown).

DISCUSSION C D The present results demonstrate that in addition to two

. I previously known major cold-shock proteins, CspA and CspB (5, 7, 10), there is another major cold-shock protein, CsdA. 4.X CsdA was found to have helix-destabilizing activity and to be a major ribosomal-associated protein at low temperature. We

* propose that CsdA plays an essential role in mRNA translation at low temperature to facilitate ribosomal function by unwinding stable secondary structures in mRNAs. It is also possible that the function of CsdA is coupled with the function of CspA, a putative RNA chaperone (6), in such a way that CspA cooperatively binds to mRNAs unwound by CsdA to prevent mRNAs from annealing. It is known that some E. coli mRNAs can contain secondary structures (for reviews see refs. 20 and F 21), which are considered to become more stable at low

. -O temperature. Since stable secondary structures in mRNAs have been shown to inhibit translation initiation (reviewed in ref. 20) as well as to pause in translation elongation reactions -O. (reviewed in ref. 21), these secondary structures are considered to be more deleterious at low temperatures. Therefore, .. the acquiring of a cold-shock-inducible helix-destabilizing protein in ribosomes becomes essential for E. coli to grow at low temperature. It is interesting to note that ribosomes have been proposed to be the physiological sensor for the cold- shock response (22) and that translation initiation factors 2a and 2f3 are also cold-shock proteins (5). In contrast to E. coli, yeast seems to constitutively contain a helicase in ribosomes whose mutation has been shown to cause a cold-sensitive FIG. 6. Effects of csdA disruption on expression of DnaK and GroEL at 15°C. Following a shift from 37 to 15°C, csd4 + (A) and csdA phenotype (reviewed in ref. 19). (B) were labeled with trans-[35S]methionine after 30 min, and csdA+ The csdA gene in multicopy suppresses the temperature- (C) and csdA (D) were also labeled after 3 hr each for 15 min. Ethanol sensitive mutation in the rpsB gene, which encodes ribosomal was added to a final concentration of 4% to other csdA + and csdA protein S2 (12). The physiological reason for the suppression cultures 30 min postshift and 2.5 hr later csdA + (E) and csdA (F) were is not known. Ribosomal protein S2 is known to be required labeled with trans-[35S]methionine for 15 min. Cellular extracts were for the binding of ribosomal protein Si to the ribosome (23). processed by two-dimensional gel electrophoresis under the same It has been demonstrated that Si protein has RNA unwinding conditions as in Fig. 1. Heat-shock proteins DnaK and GroEL are activity in the absence of ATP and is involved in the enclosed by upper and lower circles respectively, and EF-Tu is enclosed binding in a triangle. of mRNA to the 30S subunit for initiation of translation (24, 25). We have demonstrated that CsdA also has RNA unwind- respectively). It is important to note that heat-shock protein ing activity in the absence of ATP. The suppression of rpsB synthesis became derepressed in the parent strain by 3 hr of mutant by multicopies of csdA may be attributed to functional incubation at 15°C (compare Fig. 6A with 6C), while heat- complementation of ribosomal protein S1 by ribosomal pro- shock protein synthesis in the csdA4 mutant remain repressed tein CsdA. (compare Fig. 6B with 6D). Densitometry measurements using Shifting E. cOli to low temperature results in repression EF-Tu and five other major spots indicate -6-fold higher followed by derepression of heat-shock gene expression (26, synthesis of both DnaK and GroEL in the parent strain than 27). The repression was found to be at the transcriptional level in the mutant. The results indicate that the derepression of (26). The impairment of the derepression of heat-shock gene major heat shock gene expression is impaired in the mutant. expression in the csdA mutant, even in the presence of ethanol, The results obtained above suggest that the derepression of indicates the involvement of CsdA in regulation of the heat- heat-shock gene expression is specifically impaired in the csdA shock response following the shift to low temperature. The strain at 15°C. To further test whether the heat-shock response mRNA for cr32, the product of the rpoH gene that encodes the is indeed impaired in the csdA strain at low temperatures, we heat-shock transcription factor, has extensive stable secondary examined the effect of 4% ethanol on the derepression of structures (28), and the induction of or32 following a temper- heat-shock genes at 15°C. Ethanol, an inducer of the heat- ature upshift is primarily at the level of translation (28, 29). shock response (reviewed in ref. 2), was added to the csdA + Following a shift to low temperature, we propose that on the and csdA4 strains 30 mim after the shift from 37 to 15°C. As ribosome CsdA destabilizes the secondary structures of rpoH Downloaded by guest on September 26, 2021 80 Biochemistry: Jones et al. Proc. Natl. Acad. Sci. USA 93 (1996) mRNA resulting in efficient translation of the mRNA and, 8. Newkirk, K., Feng, W., Jiang, W., Tejero, R., Emerson, S. D., consequently, derepression of heat-shock gene expression. Inouye, M. & Montelione, G. (1994) Proc. Natl. Acad. Sci. USA Besides CsdA, a shift down in temperature resulted in 91, 5114-5118. increased synthesis of several ribosomal proteins (see Fig. 1). 9. Schindelin, H., Cordes, F., Jiang, W., Inouye, M. & Heinemann, Ribosomal proteins have been proposed to act as RNA U. (1994) Proc. Natl. Acad. Sci. USA 91, 5119-5123. 10. Lee, S. J., Xie, A., Jiang, W., Etchegary, J.-P., Jones, P. G. & chaperones. This was first proposed to explain the observation Inouye, M. (1994) Mol. Microbiol. 11, 833-839. that ribosomal protein S12, as well as other ribosomal proteins, 11. Yamanaka, K., Mitani, T., Ogura, T., Niki, H. & Hiraga, S. (1994) facilitated in vitro splicing of phage T4 (30). It was suggested Mol. Microbiol. 13, 301-312. that these ribosomal proteins, as RNA chaperones, mediated 12. Toone, W. M., Rudd, K. E. & Friesen, J. D. (1991) J. Bacteriol. the proper folding of the active site required for splicing (30). 173, 3291-3302. It is quite plausible at low temperature that the correct folding 13. Cosloy, S. D. & Oishi, M. (1973) Proc. Natl. Acad. Sci. USA 70, of the active conformation of certain RNA structures in vivo, 84-87. such as tRNA and rRNA, require the assistance of ribosomal 14. Wanner, B. L., Kodaira, R. & Neidhardt, F. C. (1977)J. Bacteriol. proteins including CsdA. In fact, it has been shown that some 130, 212-222. helix-destabilizing proteins can resolve misfolded tRNA and 15. VanBogelen, R. A., Hutton, M. E. & Neidhardt, F. C. (1990) 5S RNA (31, 32). Electrophoresis 11, 1131-1166. 16. Dabbs, E. R. & Wittman, H. G. (1976) Mol. Gen. Genet. 149, In addition to CsdA, E. coli contains a number of DEAD- 303-309. box proteins including SrmB, RhlB, RhlD, and DbpA (re- 17. Hardy, S. S. J., Kurkland, C. G., Voynow, P. & Mora, G. (1969) viewed in ref. 19), among which CsdA is the first prokaryotic Biochemistry 8, 2897-2905. DEAD-box protein shown to have RNA unwinding activity. 18. Flores-Rozas, H. & Hurwitz, J. (1993) J. Biol. Chem. 268, Recently, artificial overproduction of CsdA using a T7 expres- 21372-21383. sion system in E. coli has been shown to increase the stability 19. Schmid, S. R. & Linder, P. (1992) Mol. Microbiol. 6, 283-292. of certain mRNAs (33). It remains to be examined whether 20. Gold, L. and Stormo, G. (1987) in Escherichia coli and Salmonella mRNA stabilization is also a function of CsdA at low temper- typhimurium: Cellular and Molecular Biology, eds. Neidhardt, ature. F. C., Ingraham, J. L., Low, K. B., Magasanik, B., Schaechter, M. & Umbarger, E. (Am. Soc. Microbiol., Washington, DC), pp. We are grateful to Dr. B. Schwer for critical reading of the 1302-1307. manuscript. This work was supported by Grant GM19043 from the 21. Landick, R. & Yanofsky, C. 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