[Guarente, L. P., Mitchell D. H. & Beckwith, J. (1977) J. MoL. BkPL 112, 4 1 that efficient termination in vivo at the end of the trpoperon is a rho-dependent event.
Proc. Nati. Acad. Sci. USA Vol. 75, No. 11, pp. 5442-5446, November 1978
Biochemistry
Transcription termination: Nucleotide sequence at 3' end of tryptophan operon-in- Eseherichia coli (DNA sequence/dyad symmetry/RNA hairpin/rho factor)
ANNA M. WU AND TERRY PLATT Department of Molecular Biophysics and Biochemistry, Yale University, 333 Cedar Street, New Haven, Connecticut 06510
Communicated by Charles Yanofsky, August 30,1978
ABSTRACT We have determined the RNA and DNA sequences in the region specifying termination of transcription at the end of the tryptop an (t operon of Escherchia coli. A 3'-terminal mRNA fragment of about 150 nucleotides yielded oligonucleotide products that could be assigned to the end of trpA (the last structural gene in the operon) by correlation with the amino acid sequence of the protein product. Analysis of the DNA corresponding to this region served to align the few noncoding RNA oligonucleotide sequences and demonstrated that termination of tip transcription occurs in vivo at a site 36 nucleotides after tipA, with greater than 95% efficiency. In two different strains partially defective in the transcription termination factor rho, the purified transcript is much longer and more complex, sugesting that a significant amount of readthrough occurs in these strains. This is consistent with evidence [Guarente, L. P., Mitchell D. H. & Beckwith, J. (1977) J. MoL BkPL 112, 4 1 that efficient termination in vivo at the end of the trp operon is a rho-dependent event. The tp terminator (rp t) shares several features with other known sites of trgnscription termination, including (J) a 3'-terminal RNA sequence of several uridine residues, C-A-U-U-U-Uom, (ii) a GC-rich region in the DNA immediately preceding the site of termination, followed by an A-T-rich region, and (iii) a region of dyad symmetry in theDNA which, in thetranscri t,iscapableof forming a. stable hairpin containing seven GC base pairs and one AU base pair in its stem. The termination of transcription by RNA polymerase is a key event in gene expression. Termination defines the ends of genes or operons, where RNA transcripts are completed and released. Termination within genes has also been suggested as an explanation of mutational polarity (see review, ref. 1). Near the beginning of operons, termination at "attenuator" sites (2-4) may serve to control gene expression independently of the promoter or operator regions. Analysis of the primary structure of termination regions has revealed common features that may be involved in the recognition of a termination site (1). The DNA preceding a transcription termination site is often G-Grich and usually contains dyad symmetry. The RNA transcript typically ends in a series of uridine residues and can form a hairpin structure as a result of symmetry in the template. In addition, the protein factor rho (5) seems to be intimately involved in termination in vivo, although RNA polymerase can terminate at some sites in vitro in the absence of any protein factors. The tryptophan (trp) operon of Escherichia colt contains two major termination sites (Fig. la). The attenuator (trp a), a control site located before the first structural gene, causes partial termination of transcription in response to the metabolic state of the cell (2, 7, 8) and has been well characterized in vio and in vitro (for a review, see ref. 9). Transcription initiated from the trp promoter that does not terminate at trp a continues through the five structural genes to a termination site (trp t) at the end of the operon. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Early kinetic studies of tip mRNA synthesis suggested that no more than 300 nucleotides are transcribed beyond trpA (10). Detailed genetic work by Beckwith and coworkers (11, 12) subsequently showed that deletions near but not extending into trpA can place expression of distally located lac genes under trp operon control and that mutations in either rho factor or RNA polymerase can affect termination at this site (12,13). Our work identifies the 3' terminus of the 7000-nucleotide-long tip mRNA and provides the nucleotide sequence for the region specifying termination of transcription at the end of the operon. Features of this region that may signal termination and the involvement of rho factor at this site are discussed in the light of current theories concerning the mechanism of transcription termination. METHODS XtrpCBA and k8OtonB36 and an Bacteriophages Strains. E. coli strain carrying the deletion trpALD102 were the gift of C. Yanofsky. XtrpCBA carries bacterial sequences extending at least 1000 nucleotides beyond the end of the trpA structural gene. 08OtonB36 contains sequences corresponding to the carboxy-terminal 43-48 amino acids of the trpA gene product (see legend to Table 1) and extending through the adjacent tonB gene (approximately 4000 nucleotides distant). The rho201 (formerly rho tsul) and RT38 strains were generously provided by L. Guarente and J. Beckwith (13) and AD1600 carrying rho tsI5 (14) was a gift of S. Adhya. Labeling and Purification of mRNA. 32P-Labeled mRNA was prepared by the method of Bertrand et al. (15) from a strain carrying trpALD102, a trp attenuator deletion that produces high levels of distal message. Cells were starved for tryptophan and labeled with carrier-free [32P]phosphate for 25 min at 370C. After cell lysis, the RNA was purified by chloroform/methanol extraction, phenol extraction, and ethanol precipitations (16). To obtain the 3' region of tip mRNA, this material was hybridized sequentially to single-stranded DNA first from Oton86 and then from XtrpCBA (Fig. lb); mild Ti RNase treatment removed unhybridized RNA after each hybridization (17). Chromatography on Dihydroxyborylaminoethylcellulose (DBAE-Cellulose). Partial or complete T1 RNase digests of the 3' mRNA fragment were chromatographed on a 5-ml column of DBAE-cellulose (18) (gift of M. Rosenberg). Bound oligonucleotides containing a 3'-hydroxyl group were eluted by displacement with 0.1 M sorbitol (19) and ethanol precipitated once with carrier tRNA. Preparation of Restriction Endonuclease Fragments of DNA and Sequence Analysis. DNA from the transducing phage XtrpCBA was digested with the appropriate restriction endonucleases and the products were isolated by electrophoresis on polyacrylamide gels. Preparative gels contained either 3.5% or 10% acrylamide (Bio-Rad; acrylamide/bisacrylamide, 39:1) Abbreviation: DBAE-cellulose, dihydroxyborylaminoethylcellulose. 5442
Biochemistry:
Proc. Natl. Acad. Sci. USA 75 (1978)
Wu and Platt
3000 bp
4*Hin III
p,o
a
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+ trpA
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coli DNA 0 80 ton 836
-
Hinill A
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5443
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I
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3' mRNA fragment
C
DNA
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234 l
'
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I
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t
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Hpal
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-
FIG. 1. (a) The tryptophan operon in E. coli. We have adopted the convention of using capitals for structural genes and lower case for regulatory elements: E, D, C, B, and A, structural genes for tryptophan biosynthetic enzymes; p, promoter; o, operator; a, attenuator; t, terminator. The two Hin III sites shown define a 3000-base-pair fragment of E. coli trp DNA (6), which is the smallest product of Hin III digestion of XtrpCBA DNA. (b) Hybridization scheme for isolation of a 150-nucleotide-long 3'-terminal fragment of trp mRNA. Formally, the first hybridization against 080tonB36 DNA should be sufficient; however, we find that a second hybridization against XtrpCBA DNA (which contains much less distal sequence) is necessary to remove contaminating tonB sequences. The numbering refers to amino acid residue positions in the trpA gene product. (c) Restriction endonuclease cleavage sites in the terminator region. Recognition sequences (5' 3') are: Hin III, A-A-G-C-T-T; Hae III, G-G-C-C; Taq I, T-C-G-A; Tac I, C-G-C-G; Hha I, G-C-G-C; Hpa II, C-C-G-G. Black areas indicate the extent of each DNA strand that was sequenced. -
in 50 mM Tris borate, pH 8.3/1 mM EDTA. Fragments were eluted from gel slices by electrophoresis in 40 mM Tris acetate, pH 8.5. The 3000-base-pair Hin III fragment (Fig. la) was isolated by sedimentation on 5-20% sucrose gradients in 1 M NaCI/10 mM Tris, pH 7.6/1 mM EDTA in a Beckman SW 50.1 rotor for 4 hr at 48,000 rpm at 4°C. After treatment with bacterial alkaline phosphatase (gift of J. Chlebowski), DNA fragments were end-labeled with ['y32P]ATP (3000 Ci/mmol, New England Nuclear) by using T4 polynucleotide kinase (P-L Biochemicals). Mapping of restriction endonuclease cleavage sites within the Hin III fragment and near the terminator region was carried out by the method of Smith and Birnstiel (20). DNA sequences were determined by using the chemical cleavage method of Maxam
and Gilbert (21). Restriction endonucleases Hpa II, Hin II, Hin III, Hha I, Taq I, and Hae III were purchased from New England BioLabs. Hpa II was also obtained from N. Grindley, Hae III from S. Weissman, and Taq I from R. Young.
RESULTS 3' Region of tip RNA. A fragment of mRNA derived from the 3' region of the trp operon was isolated by hybridization of in vivo labeled [32P]mRNA to DNA from two different t-rp transducing phage, as indicated schematically in Fig. lb. A two-dimensional fingerprint of the Ti RNase digestion products of the purified RNA (Fig. 2a) suggests a size of approximately 150 nucleotides, in agreement with estimates obtained by electrophoresis of the fragment on 10% polyacrylamide/7 M urea gels with trp leader mRNA (140 nucleotides) as a size marker (not shown). Eluted Ti oligonucleotides were further analyzed by using pancreatic RNase, U2 RNase, carbodiimide modification, and alkaline hydrolysis according to standard procedures (22). The final sequences of the Ti oligonucleotides are presented in Table 1. The pancreatic RNase digestion products of the purified mRNA fragment provided overlap information and were consistent with the overall sequence (data not shown).
Most of the Ti oligonucleotide sequences could be aligned with the carboxy-terminal amino acid sequence of the trpA gene product, the a-subunit of tryptophan synthetase. However, only oligonucleotides 13, 19, and 22 could not be placed in the translated portion of the sequence. When a partial Ti Table 1. T1 oligonucleotides from 3' end of trp mRNA
Sequence
Location
G C-G A-G C-C-G C-A-G/A-C-G C-U-G A-U-G 262 C-C-A-G 248 A-A-A-G 256,263 U-U-C-G (234) A-U-U-G (224) C-A-C-U-G 254 U-U-C-C-G U-U-U-U-U-G 257 A-A-A-A-U-G 250 C-C-A-U-U-G 236 A-U-U-U-C-U-G (232) U-A-C-A-A-C-C-G 259 U-U-A-A-U-C-C-C-A-C-A-G 268 U-U-A-A-A-A-U-C-A-U-C-G 238 C-A-A-C-A-U-A-U-U-A-A-U-G 243 C-A-U-U-U-UoH The oligonucleotides are numbered to correspond to their positions on the wild-type (rho+) fingerprint (Fig. 2); "location" refers to amino acid position in the trpA protein (see Fig. 4). Oligonucleotides 10, 11, and 17 can be aligned with the protein sequence between residues 224 and 235 but the assignments have not been confirmed by DNA sequence analysis. The RNA data indicate that the end point of bacterial sequences in ,80tonB36 occurs in a region corresponding to amino acids 220-224 of the trpA protein. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Biochemistry: Wu and Platt
5444
Proc. Natl. Acad.- Sci. USA 75 (1978)
a
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FIG. 2. Ti RNase fingerprints of 32P-labeled trp mRNA. The purified RNA was digested and analyzed as described by Squires et al. (16). (a) "Wild-type" 3'-terminal RNA isolated from a strain carrying trpALDlO2 as outlined in Fig. lb. For location and sequences of these oligonucleotides see Table 1 and Fig. 3. Oligonucleotide 22 is retained on a DBAE-cellulose column (see text) and represents the 3' terminus, C-A-U-U-U-UOH. Its unusual mobility in the chromatographic direction is expected for an oligonucleotide with a 3'-terminal OH (15). (b) mRNA isolated from a rho2OW strain by the same hybridization scheme as in a. The complexity of this fingerprint suggests that read-through is occurring at trp t (see text).
digest of the RNA was passed through a DBAE-cellulose column (which selectively retains RNA sequences containing a 3'hydroxyl group), the sorbitol-eluted material yielded a T1 fingerprint enriched in the large oligonucleotides 13, 19, and 22, suggesting that they appear close to the 3' end of the
mRNA.
Identification of 3'-Terminal Sequence. When a complete T1 RNase digest of the RNA fragment was chromatographed on a DBAE-cellulose column, only oligonucleotide 22 was bound. The molar yield of this oligonucleotide from the fingerprint was consistently 80-100%. Other 3'-terminal oligonucleotides would have been detectable at the 10% level, but none was evident. The sequence of oligonucleotide 22, C-AU-U-U-UOH, was deduced from the following information. Digestion with pancreatic RNase yielded A-U, C, and U but no products containing G. Alkaline hydrolysis verified the absence of G. U2 RNase digestion yielded C-A plus a larger product containing neither A nor G. This product is probably U-U-UUOH because it yielded only Up after alkaline hydrolysis and pU upon complete digestion with snake venom phosphodiesterase, and its electrophoretic mobility did not change after treatment with bacterial alkaline phosphatase. Its mobility on DEAE paper at pH 3.5 was slightly higher than that of the marker U-U-U-GOH, implying a content of three phosphates. All the above data, and the anomalous mobility of oligonucleotide 22 in the two-dimensional separation, are consistent with the unique 3'-terminal sequence C-A-U-U-U-UOH for trp operon
mRNA.
DNA Sequence across the Termination Region. DNA from the bacteriophage XtrpCBA was the source of fragments spanning the termination region of the trp operon. Initially, we derived the map of restriction endonuclease cleavage sites as shown in Fig. ic. The DNA sequence of a 150-nucleotide-long region extending from a Hae III site at alanine-236 to a Hpa II site 55 nucleotides beyond the end of the structural gene was then determined from 5'-terminal labeled fragments by the technique of Maxam and Gilbert (21). For the translated region, only one strand of DNA was sequenced (see Fig. ic); however, the sequence agreed completely with the RNA sequence obtained independently and with the protein sequence (Fig. 3). For distal sequences, both strands were analyzed (Fig. ic), although some problems were encountered on one strand in and beyond the termination region, due to secondary structure artifacts on the gel. The DNA sequence specified the order of the RNA oligonucleotide sequences outside trpA and extended the total sequence information 20 nucleotides beyond the 3' end of the RNA sequence. rho Dependence of the trp Terminator. Guarente et al. (12) reported that rho2Ol, a mutation in the gene for rho termination factor, permits read-through at trp t. To test this directly, we purified the mRNA from a strain carrying rho201 and compared it to that obtained from an isogeneic wild-type strain. The complex fingerprint of rho201 mRNA (Fig. 2b) indicates the presence of oligonucleotide sequences beyond the trp operon, suggesting that read-through does occur. However, the normal terminal oligonucleotide 22 is also detectable in this strain and hence termination must still be occurring at trp t at perhaps 20-40% of the usual efficiency. The predicted readthrough oligonucleotide, C-A-U4-A-A-C-U3-C-U3-A-A-U-G, has not yet been identified. mRNA prepared from AD1600, a strain carrying the rho ts15 mutation (14), yielded a fingerprint similar to the rho201 fingerprint, indicating that read-through at trp t also occurs with this rho mutation. DISCUSSION We report here the nucleotide sequence of a region specifying termination of-transcription at the end of a bacterial operon. In E. coli, the 7000-nucleotide-long mRNA of the trp operon ends at a unique point 36 nucleotides beyond the final gene. Determination of this end point required isolation of the 3' region of the mRNA and alignment of the RNA sequence with the DNA sequence of this region. An unambiguous primary structure for the remainder of the transcribed region was derived by using both RNA and DNA sequence information and was, in turn, consistent with protein sequence in the translated portion. The interesting features of the termination region are shown in Figs. 3 and 4. First, the untranslated RNA is relatively small, much shorter than the 90 nucleotides of noncoding RNA beyond the end of the cro gene in bacteriophage X (26). Second, the transcript has a terminal sequence of four uridine residues. Many 3'-terminal sequences of RNA in prokaryotic organisms also have U-rich termini (1). Third, a center of dyad symmetry (shown in Fig. 3) is located 21 nucleotides beyond trpA, 15 nucleotides before the site of transcription termination. In the RNA transcript, this symmetry would permit intramolecular base-pairing and formation of an RNA hairpin containing seven G-C base pairs and one A-U base pair in its stem (Fig. 4). The AG of this structure is about -21 kcal (1 kcal = 4.18 kJ)/mol, if calculated according to the rules of Gralla and Crothers (27). The dyad symmetry and the potential for stable hairpin formation are common features in regions of transcription termination. Fourth, G-C base pairs predominate in the untranslated region, comprising 75% of the 28 nucleotide pairs from
Biochemistry:
Proc. Natl. Acad. Sci. USA 75 (1978)
Wu and Platt 235
240
245
250
5445
255
SER-ALA-ILE-VAL-LYS-ILE-ILE-GLU-GLN-HIS-ILE-ASN-GLU-PRO-GLU-LYS-MET-LEU-ALA-ALA-LEU-LYS-
trpA protein mRNA sequence
T1
5'
oligonucleotides
DNA
Restriction endonuclease cleavage sites
5' 3'
GPG
C GGC AUU GUU AAA AUC AUC GAG CGM CAU AUU AAU GAG CCA 16 20 21 8
AAA AUG.CUG GCG GCA CUG AAA 12 15 9
G GCC ATT GTT AAA ATC ATC GAG CAA CAT ATT AAT GAG CCA GAG AAA ATG CTG GCG GCA CTG AAA C CGG TAA CAA TTT TAG TAG CTC GTT GTA TAA TTA CTC GGT CTC TTT TAC GAC CGC CGT GAC TTT Hae III Taq I
265
260
268
VAL-PHE-VAL-GLN-PRO-MET-LYS-ALA-ALA-THR-ARG-SER GUU UUU GUA CAA CCG AUG AAA GCG GCG ACG CGC AGU UAAUCCCACAGCCGCCAGLTUCCGCUGGCGGCAUUUU 18 7 9 19 5 8 13 14 6 22
3'
GTT TTT GTA CAA CCG ATG AAA GCG GCG ACG CGC AGT TAATCCCACAGCCGCCAGTTCCGCTGGCGGCATTTTAACTTTCTTTAATGAAGCCGG CAA AAA CAT GTT GGC TAC TTT CGC CGC TGC GCG TCA ATTAGGGTGTCGGCGGTCAAGGCGACCGCCGTAAAATTGAAAGAAATTACTTCGGCC Tac I 1 20 30 40 50 Hpa II 10 Hha I AT-rich GC-rich
FIG. 3. Nucleotide sequence of the termination region of the trp operon, indicating alignment of protein, RNA, and DNA sequences. The numbers below the RNA sequence identify the RNase T1 oligonucleotides (Table 1). The numbers below the DNA sequence indicate the number of base pairs following the structural gene. The region of dyad symmetry is indicated with bars. In the protein sequence, the assignment of Ile-Asn (rather than Asn-Ile) for amino acids 245-246 (23) is the correct one. Because nonsense codons in the other reading frames of this region preclude the translation required for the read-through trpA protein of Hardman et al. (24), it seems more likely that their mutant protein is due to an alteration in the DNA other than a simple frameshift mutation. Features of unknown importance include a 10-base-pair direct repeat (dashed lines), and an 8-base-pair inverted repeat (dotted lines).
the ochre codon through the symmetric region preceding the terminator. In contrast, 18 of the next 21 base pairs are A-T. As discussed below, such a skew in G-C/A-T distribution is seen in many (although not all) termination regions. In these respects, the trp terminator region resembles the trp attenuator region (see ref. 25) as illustrated in Fig. 4. Most of the other currently known termination regions also include a U-rich 3' terminus (1). However, in both the X TRI transcript (26) and that of the tRNATYr genes (28), which are rho-dependent sites in vitro, the U-rich terminus is curiously lacking. Another common feature of termination regions is a G-C-rich region present immediately before the uridine residues, and Gilbert (29) has hypothesized that such a strongly base-paired region may enhance termination. Exceptions to this are again X TRI with few G-Cs anywhere in the termination region and the tRNATYr terminator with a concentration of
G.Cs just beyond the site of termination (28). In these cases, and in the X 4S and 6S transcripts (30), there are no obvious homologies in the actual nucleotide sequences distal to the point of termination. However, in the leader region of the trp operon, the deletion trpALCl419 removes part of the A-T-rich sequence distal to the termination site and eliminates four of the eight terminal uridine residues in the transcript. Bertrand et al. (15) found that, with a strain carrying this deletion, termination in the attenuator region is only partially effective in vivo and absent in vitro; therefore, some portion of the deleted sequences must be essential for complete function. The influence of secondary structure on termination must also be considered, because significant dyad symmetry in the transcribed region has been observed in all cases thus far except for the tRNATYr terminator. A stable structure in the RNA could interact directly with polymerase and termination factors or
A.AU
b
uI
U
/
A- U C*G
G- C G- G CoG C- G
Ge C 5' CAGAUACCC A UUUUUUUOH 3
(a) ATTENUATOR SITE
G
C- G
5'
UA AUCCC AC AG
AU UUUOH 3'
(b) TERMINATOR SITE
FIG. 4. Stable hairpin structures that can be formed by trp mRNA at the attenuator (a) and the terminator (b). Lee and Yanofsky (25) have proposed that the equilibrium between the leader mRNA structure shown and an alternate structure (not shown) may be shifted by translation of the leader mRNA to regulate the extent of attenuation. In the terminal region of trp mRNA, a secondary structure that competes with the hairpin shown is conceivable (utilizing the 10-base repeat shown in Fig. 3), but we have no specific evidence that either structure forms.
5446
Biochemistry: Wu and Platt
could simply facilitate release of the completed RNA molecule from the DNA template to which it has been base paired. Support for the role of RNA secondary structure in termination function at the trp attenuator is provided by a comparison of the E. coli sequence with that of Salmonella typhimurium (25, 31). The predicted stability of the secondary structures correlates with the efficiency of termination in vitro in each case. At the bacteriophage X termination site TRI, point mutations that reduce the dyad symmetry (even though increasing the G-C content of the region) have been shown to reduce the efficiency of termination (26). Mutations affecting termination at the end of the trp operon have been examined by Beckwith and coworkers (12, 13). Two classes of mutations unlinked to the trp operon were found, those in RNA polymerase (rpo2O3) and those in rho factor (rho201). Guarente et al. (12) have shown that in the presence of rho201 the expression of distally placed lac genes was subject to trp operon control. These results suggested that RNA polymerase was unable to terminate with normal efficiency at trp t and continued transcription into the lac operon. Our data support this interpretation by showing that rho2Ol does, in fact, permit considerable read-through at trp t. Residual termination activity, as measured by the yield of oligonucleotide 22 (Fig. 2), is less than 40% of the normal level. Similar results with the rhotsl5 mutation (data not shown) indicate that, in vivo, efficient termination of transcription at the end of the trp operon requires functional rho factor. Guarente et al. (12) have demonstrated the existence of a third class of mutations with linkage to the trp operon which suggest alterations in the terminator region itself. Many of these mutations are deletions of various lengths (150-700 base pairs) immediately distal to trpA (13). Our preliminary characterization of one of these, RT38, indicates that it is a deletion of 220 base pairs, beginning between 20 and 75 base pairs distal to the point of termination of transcription (unpublished data). Present evidence does not allow us to distinguish between effects on termination and new initiations of transcription enhanced by the presence of the deletion. We also cannot rule out the possibility that the observed 3' end is the result of processing that is dependent on a downstream termination event. However, no distal sequences are detected by our technique, and thus this model must include rapid degradation of the processed fragments. Overall, it is clear that analysis and evaluation of the signals required for termination are quite complex. There may be a considerable difference between termination at rho-dependent sites compared to that at rho-independent sites, and the behavior of RNA polymerase in purified systems in vitro is not always the same as it is in vivo (1). Nor is it understood whether the signal causing termination is conveyed in the primary base sequence or the secondary structure of the DNA or its RNA transcript. Indeed, both sequence and structure may be involved to varying degrees depending on the presence of additional factors such as rho (32), X N protein (1), or att factor (33). We thank Charles Yanofsky, Jon Beckwith, and Leonard Guarente for strains and helpful advice. This work was supported by U.S. Public Health Service Grant GM22830 to T.P.; A.M.W. was a predoctoral trainee on U.S. Public Health Service Grant GM07223.
Proc. Nati. Acad. Sci. USA 75 (1978) 1. Adhya, S. & Gottesman, M. (1978) Annt. Rev. Biochem. 47, 967-996. 2. Bertrand, K., Korn, L., Lee, F., Platt, T., Squires, C. L., Squires, C. & Yanofsky, C. (1975) Science, 189,22-26. 3. Yanofsky, C. (1976) in Molecular Mechanisms in the Control of Gene Expression, ICN-UCLA Symposia on Molecular and Cellular Biology, eds. Nierlich, D., Rutter, W. J. & Fox, C. F. (Academic, New York), Vol. 5, pp. 75-87. 4. Winkler, M. E., Roth, D. J. & Hartman, P. E. (1978) J. Bacteriol. 133,830-843. 5. Roberts, J. (1969) Nature (London) 224, 1168-1174. 6. Hopkins, A. S., Murray, N. E. & Brammar, W. J. (1976) J. Mol. Biol. 107, 549-569. 7. Morse, D. E. & Morse, A. N. C. (1976) J. Mol. Biol. 103,209226. 8. Bertrand, K. & Yanofsky, C. (1976) J. Mol. Biol. 103, 339349. 9. Platt, T. (1978) in Molecular Aspects of Operon Control, eds. Miller, J. H. & Reznikoff, W. S. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), in press. 10. Rose, J. K. & Yanofsky, C. (1971) J. Bacteriol. 108,615-618. 11. Mitchell, D. H., Reznikoff, W. S. & Beckwith, J. (1976) J. Mol. Biol. 101,441-457. 12. Guarente, L. P., Mitchell, D. H. & Beckwith, J. (1977) J. Mol. Biol. 112,423-436. 13. -Guarente, L. P. & Beckwith, J. (1978) Proc. Natl. Acad. Sci. USA 75,294-297. 14. Das, A., Court, D. & Adhya, S. (1976) Proc. Natl. Acad. Sci. USA 73, 1959-1963. 15. Bertrand, K., Korn, L. J., Lee, F. & Yanofsky, C. (1977) J. Mol. Blot. 117,227-247. 16. Squires, C., Lee, F., Bertrand, K., Squires, C. L., Bronson, M. J. & Yanofsky, C. (1976) J. Mol. Biol. 103,351-381. 17. Platt, T. & Yanofsky, C. (1975) Proc. Natl. Acad. Sci. USA 72, 2399-2403. 18. Rosenberg, M. (1974) Nucleic Acids Res. 1, 653-671. 19. Rosenberg, M., Weissman, S. & deCrombrugghe, B. (1975) J. Biol. Chem. 250, 4755-4764. 20. Smith, H. 0. & Birnstiel, M. L. (1976) Nucleic Acids Res. 3, 2387-2398. 21. Maxam, A. M. & Gilbert, W. (1977) Proc. Natl. Acad. Sci. USA 74,560-564. 22. Brownlee, G. G. (1972) Determination of Sequences in RNA (North-Holland/American Elsevier, New York). 23. Guest, J. R., Carlton, B. C. & Yanofsky, C. (1967) J. Biol. Chem. 242,5397-5412.
24. Hardman, J. K., Berger, H. & Goodman, M. (1975) J. Biol. Chem. 250,4634-4642. 25. Lee, F. & Yanofsky, C. (1977) Proc. Natl. Acad. Sci. USA 74, 4365-4369. 26. Rosenberg, M., Court, D., Wulff, D. L., Shimatake, H. & Brady, C. (1978) Nature (London) 272,414-432. 27. Gralla, J. & Crothers, D. M. (1973) J. Mol. Biol. 73, 497-511. 28. Kupper, H., Sekiya, T., Rosenberg, M., Egan, J. & Landy, A. (1978) Nature (London) 272,423-428. 29. Gilbert, W. (1976) in RNA Polymerase, eds. Losick, R. & Chamberlin, M. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), pp. 193-205. 30. Rosenberg, M., deCrombrugghe, B. & Musso, R. (1976) Proc. Natl. Acad. Sci. USA 73, 717-721. 31. Lee, F., Bertrand, K., Bennett, G. & Yanofsky, C. (1978) J. Mol. Biol. 121, 193-217. 32. Roberts, J. W. (1976) in RNA Polymerase, eds. Losick, R. & Chamberlin, M. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), pp. 247-271. 33. Pouwels, P. H. & vanRotterdam, J. (1975) Mol. Gen. Genet. 136, 215-226.
