Molecular Microbiology (1992) 6(19), 2769-2776

Perturbing highly conserved spatial relationships in the regulatory domain that controls inducible cat translation Zhjping Gu^ and Paul S. ^Department of Biological Sciences, University of Maryland, Catonsville, Maryland 21228. USA. ^The Center for Marine Biotechnology, Maryland Biotechnology Institute, Baltimore, Maryland 21201, USA.

Summary ChloramphenJcol activates translation of cat-86 mRNA by stalling a ribosome in the leader of individual transcripts. Stalling triggers two sequential events: the destabilizatJon of a region of secondary structure that sequesters the cat ribosome-binding site (RBS-C), and the initiation of caf translation. The site of drug-dependent ribosome stalling is dictated by the leader sequence, crb; crb causes a ribosome to stall with its aminoacyl site at leader codon 6. We demonstrate that induction requires the maintenance of a precise spatial relationship between crb and sequences within the left inverted repeat of the secondary structure. Therefore, destabilization of the secondary structure during chloramphenicol induction may result from the interaction of a stalled ribosome with a specific sequence in the secondary structure rather than from non-specific masking of RNA sequences, cat-86 regulation also depends on the distance that separates crb from RBS-C. This interval of 33 nucleotides was incrementally increased and decreased by mutations within a loop in the secondary structure. Shortening the distance between crb and RBS-C by three nucleotides reduced induction by half and a deletion of nine nucleotides abolished induction. Insertion mutations were without effect on induced expression but elevated basal expression. The results indicate that when the A site of a ribosome occupies leader codon 6 the secondary structure is destabilized and there is no interference with entry of a second ribosome at RBS-C. The data further demonstrate that when the A site of a ribosome in the leader is within 30 nucleotides of RBS-C, cat

Received8April, 1992; revised4 June, 1992; accepted 15 June, 1992, 'For con-espondence. Tel. (410) 455 2249; Fax (410) 455 3875.

expression decreases. This decrease probably results from competition of the leader ribosome with the ribosome initiating cat translation. Our observations demonstrate that in wild-type cat-86 the distances between crb and the secondary structure, and between crb and RBS-C provide the precise spacing necessary to achieve three interdependent effects: the destabilization of the RNA secondary structure by a ribosome stalled at crb; a lack of competition between a ribosome stalled at crb and the initiating ribosome; and maintenance of a low, but measurable, basal level of cat expression. The spatial relationships identified as necessary for the regulation of cBt-86 are conserved in the regulatory regions for five other inducible cat genes.

Introduction Translation attenuation is a genetic regulatory device that functions by modulating mRNA translation {Bruckner and Matzura, 1985; Dubnau, 1984; Duval and Lovett, 1986; Lovett, 1990; Weisblum, 1983). Genes under this form of control encode transcripts that are normally untranslated because of sequestering of the ribosome-binding site in secondary structure. The negative regulation imposed by the RNA secondary structure can be relieved by stalling a ribosome in an upstream region of the transcripts, i.e. the leader. The chemical that brings about the required ribosome stalling is the inducer, and the two types of genes proposed to be regulated by translation attenuation have specific leader sequences enabling them to respond to different inducers. erm genes, which are regulated by translation attenuation, are induced by erythromycin, although some erm genes can also be induced by other macrolide antibiotics (Weisblum, 1983). Inducible cat genes are regulated by translation attenuation and chloramphenicol is the inducer; one example gene, cat-86, also can be induced by the nucleoside antibiotic amicetin and by erythromycin (Duvall et al., 1985; Rogers and Lovett, 1990; Lovett, 1990). erm and ca( genes confer resistance to erythromycin and chloramphenicol, respectively, and hence the classical inducers of these genes, erythromycin and chloramphenicol, are the same antibiotics to which the genes confer resistance.

2770 Z. GuandP. S. Lovett Fig. 1. Conserved spatial relationships within the regulatory domain of chloramphenicol-inducible cat genes. RBS-L and R8S-C are the ribosomebinding sites for the leader and cat structural genes. Data for genes cat-86. cat-221, cat-57. cat-66. cat-194. and cat-112, with which this diagram was constructed, are summarized by Duvall etai (1987). Primary sequence data are found in Ambulos et ai (1986), Bruckner and Matzura (1985), Duvall etai (1983). Duvall etai. (1984), Horinouchi and Weisblum (1982). and Shaw e( a/. (1985).




—V/////. CODON 6

Ribosome stalling in the leader is, in itself, sufficient for cat-86 induction as demonstrated by activation of gene expression through the stalling of a drug-free ribosome in the leader by amino acid starvation (Duvall et ai, 1987). Expression is detected only when the aminoacyl (A) site of a ribosome is stalled within a very specific region of the leader (Alexieva et ai, 1988). Maximum expression is obtained when the A site is stalled at leader codon 6, located at the 5' base of the RNA secondary structure. Stalling at leader codons 7 or 8, Vifhich are within the secondary structure, yields weak cat-86 expression and stalling at leader codons 3, 4 or 5 fails to induce the gene. Leader codon 6, therefore, marks the location of the A site of a stalled ribosome that is most active in the induction. The site of ribosome stalling due to the action of chloramphenicol also causes the A site of the ribosome to occupy leader codon 6 {Alexieva etai, 1988). However, in the case of drug induction, the site of stalling depends on, and is dictated by, the leader sequence, crb (Rogers et ai, 1990b). The mechanism through which crb contributes to the specificity of stalling remains unclear although crb has been shown to share extensive complementarity with an internal sequence of 16S rRNA (Rogers et ai, 1990a). Thus, it is possible that a crb-rRNA pairing may aid in the precise placement of a ribosome during drug-mediated stalling. It is thought that a ribosome stalled at leader codon 6 activates cat-86 expression by destabilizing the RNA secondary structure, which frees the cat-86 ribosomebinding site and permits translation initiation. The favoured model for induction therefore involves two ribosomes: one ribosome stalled in the leader, and a second ribosome that initiates translation at the ribosome-binding site for the cat structural gene. A prokaryotic ribosome

reportedly spans (protects) 35 to 40 nuoleotides of mRNA (Gold et ai, 1981). Consequently, the two-ribosome model places constraints on the minimum distance that must exist between the site of ribosome stalling in the leader and the location of the initiating ribosome. Here we demonstrate two spatial relationships which are necessary to the observed regulation of cat-86. The first is the relationship between crb and the secondary structure, and the second is the relationship between a ribosome stalled at crb and a ribosome initiating translation of cat. Our results reveal that the precise positioning of both the stalled and initiating ribosomes is necessary for translation attenuation to be an effective form of regulation for ca^ gene expression. Results A spatiai reiationship essential to cat induction: the location of crb relative to the left inverted repeat cat-86 leader codons 2 to 5, designated crb, and chloramphenicol cause a ribosome to stall in the leader with its aminoacyl (A) site at leader codon 6 (Rogers etai, 1990b). The distances from crb to three downstream sites are conserved among the six inducible cat genes for which sequence data are available: seven nucieotides (nt) separate crb from anti-RBS-C; 17 nt separate crb from the end of the left inverted repeat; and 33-36 nt separate crb from RBS-C (Fig. 1). It has been shown that drug-induction of cat-86 is lost when crb is moved away from the left inverted repeat by inserting an extra codon (codon 5A) between leader codons 5 and 6 (Rogers et ai, 1990b). Restoration of full inducibility is then obtained by deleting leader codon 6 (Rogers etai, 1990b). The deletion causes

cat gene regutation 2771

1 2 3 4 5 6 7 8 Mcl Val Lys Thr Asp Lys [le Sei Scr





Leader —

Regulating Ribosome

I n i t i a t i n g Ribosome

Fig. 2, Model for the positioning of the regulating and initiating ribosomes during chloramphenico) induction of cat-86. The regulating ribosome is stalled with its A site at leader codon 6. The P site therefore occupies leader codon 5. For this diagram it has been assumed that a ribosome spans (contacts) 40 nt of mRNA and that the contact region extends 12 nt 3' to the A site (see Gold and Stormo, 1987). The secondary structure shown in Fig. 1 is represented here as a linear form that might arise during destabilization.

codon 5A (Asp) to occupy the position normally occupied by a Lys codon. To determine the effect of moving crb closer to the left inverted repeat, leader codon 6 was deleted from wild-type cat'86. This mutation also prevented drug-induction (Table 1). To determine if the loss of inducibility was due to the proximity of orb to the left inverted repeat or to sequences further downstream, three nt were inserted into the loop of a version of cat-86 lacking leader codon 6. The -i-3 loop insertion did not restore inducibility (Table 1). Thus, the essential relationship is the proximity of crb to the left inverted repeat, or to sequences contained within the left inverted repeat such as anti-RBSC.

series of deletion and insertion mutations was made in the loop region of the RNA secondary structure. Deletion of 3, 6 and 9 nt from the loop diminished induced expression by approximately 50%. 85% and >99%, respectively, whereas loop insertions of up to 13 nt had no measurable effect on Induction (Fig. 3A). These data suggest that in wild-type cat'86 the distance from crb to RBS-C is adequate to prevent interference between the stalled and initiating ribosomes. Shortening the intervening distance by as little as 3 nt reduces cat-86 expression which is probably due to interference with cat translation initiation.

The nature of leader codon 6 seems relatively unimportant for cat-86 induction as judged from studies in which this codon was changed from Lys to a Tyr or an Asp codon. Rather, leader codon 6 appears primarily to provide the correct spacing between crb and sequences contained within the left inverted repeat. For example, crb may be positioned to stall a ribosome at the appropriate location to facilitate a second interaction with the left inverted repeat, resulting in localized melting of the secondary structure.

Loop insertion mutations etevate basat expression of oat'86

Effect on induced cat-86 expression of detetions and insertions in the toop Published values for the positioning and space occupied by a prokaryotic ribosome (Gold et ai, 1981; Gold and Stormo, 1987) suggest that a ribosome stalled at crb, with its A site at leader codon 6, should not interfere with translation initiation by a second ribosome at RBS-C (Fig. 2). However, the region of mRNA occupied by a single ribosome has typically been inferred from the number of nt (35-40) which are ribosomally protected from agents such as RNase or dimethylsulphate. It is not unreasonable to believe that the physical presence of a ribosome extends beyond the protected nucleotides. To test this idea, a

The basal (uninduced) level of cat-86 expression is quite high, amounting to 5-10% of induced expression (e.g. Alexieva etat., 1988). Mutations that prevent a ribosome from translating beyond leader codon 5 reduce basal expression by a factor of 5-10. This result has been obtained both with cat-86 and cat-112 (Alexieva et at., 1988; Bruckner ef ai, 1987). It is therefore likely that basal expression is due to a ribosome translating into the secondary structure. This causes a transient destabilization which can permit occasional ribosome loading at RBS-C and, consequently, low-level caf expression.

Table 1, Effect of deleting leader codon 6 on the induction of cat-86 by chloramphenicol.^ CAT-specific Activity Gene



cat-86 wild type

0.54 0.28 0.40 1.1

6.2 0.31 0.41 6.7

cat-86 M6 cat-86 AL6 +3LP cat-86 +3LP

a. AL6 is the deletion of leader codon 6 and + 3LP is the insertion of 3 nt into the loop (see the Experimental procedures).

2772 Z Gu and P. S. Lovett Fig. 3. Induced (A) and basal (B) expression of mutants of cat-86 containing deletions and insertions in the loop. The construction of these mutants is described in the Experimental procedures. 'Zero nts' is the wild-type gene containing a 12 nt loop. Induced values of CAT are corrected for basal expression. Thus, the actual CAT-specific activity observed after induction of each plasmid-containing strain is the sum of the values shown in A and B. Hence, the induced CAT-specific activity observed for the +10 insertion mutant is 7.3; 5.2 (from A) plus 2.1 (panel B).









Nts Deleted or Added to Loop

Deletions from the loop reduced basal expression in parallel with the reduction in induced expression (Fig. 3), This result was expected and is consistent with the suggestion that a ribosome at leader codon 6 must remain at least 30 nt from RBS-C to avoid competing with translation initiation of cat. On the other hand, loop insertion mutations elevated basal expression, and the increase was proportional to the number of nt added to the loop (Fig. 3B). We propose that the increase in basal expression is largely due to increasing the number of leader codons within the secondary structure that are 30 nt or more from RBS-C. For instance, in wild-type cat-86, leader codon 7 is 27 nt from RBS-C (Fig. 2), and a ribosome at this codon would probably compete with translation initiation based on data shown earlier. By inserting 3 nt into the loop, leader codon 7 becomes 30 nt from RBS-C and a ribosome at codon 7 should not interfere with caf translation initiation. Each leader codon at which a ribosome can destabilize the secondary structure but not interfere with cat translation initiation contributes to basal expression. In wild-type oat-86, only leader codon 6 fulfils both requirements. In the +3 loop insertion mutant codons 6 and 7 fulfil the requirements, etc. Increasing the number of leader codons that fulfil both requirements will increase the time, during leader translation, for which RBS-C is available for translation initiation; hence the increase in basal expression due to loop insertion mutations. The above interpretation presumes that the increase in basal expression due to the loop insertion mutations was not an artifact resulting from an unstable secondary structure. To test this, leader codon 4 of the +^0 loop insertion mutant was mutated to an ochre codon. The result was a 10-fold reduction in basal expression, indicating that high basal expression required translation into the secondary structure (Table 2).

Additionally, leader codon 4 (Thr; ACA) of the +10 loop insertion mutant was changed to the proline codon CCA. The mutation to a proline codon has previously been shown to prevent chloramphenicol induction of both cat-112(Dick and Matzura, 1988) and cat-86{E. J. Rogers, unpublished). Although the proline codon prevented induction of the -t-10 loop insertion mutant, basal expression remained high (Table 2). Thus, the elevation in basal expression due to loop insertion mutations required translation into the secondary structure, but did not require a leader sequence that was capable of supporting induction by chloramphenicol.

Discussion The present study identifies two interdependent spatial relationships within the regulatory domain of caf-S6 transcripts which are necessary for the activation of cat translation by chloramphenicol. The first involves the positioning of the stall sequence crb relative to sequences of the lett inverted repeat. Changing the spacing between these elements by adding or deleting three intervening nt abolished induction. This essential relationship could be necessary for site-specific, drug-dependent ribosome

Table 2. Effect of changing leader codon 4 to the ochre codon (TAA) or the Pro codon (CCA) on basal and induced expression ot the +10 loop insertion mutant of cat'86. CAT-specific Activity Gene



cat-86 wild type cat-86+10LP oat-86 +10LPL4TAA cat-86 +10LPL4CCA

0.49 2.3 0.18 2.4

6.3 7.3 0.14 2,5

cat gene regulation


stalling, or for destabilization of the secondary structure or possibly both. However, moving crb away from the left inverted repeat did not perturb induction brought about by starving cells for the amino acid specified by the codon at the 5' base of the secondary structure {Rogers ef ai, 1990b), a process that clearly depends on destabilization of the secondary structure. Thus, it is likely that the proximity of crb to the left inverted repeat enables chloramphenicol to place a ribosome stably at the induction site. For example, chtoramphenicol may cause transient ribosome pausing at crb which is subsequently converted to stable ribosome stalling through a second interaction of the ribosome with anti-RBS-C located precisely 7 nt downstream of crb. It is conceivable that the recognition by a ribosome of crb and relevant sequences in the left inverted repeat is through complementary pairing of sequences in rRNA and mRNA (Dahlberg, 1989; Rogers efa/., 1990a; Green efa/., 1985).

RBS-C, by loop insertion mutations, did not enhance inducibie expression over that seen with the wild-type interval of 33 nt. However, the loop insertions did have a striking effect on basal expression, causing an increase in basal expression that was proportional to the number of nucleotides inserted into the loop. This observation suggests that basal expression, unlike induced expression, is probably due to non-specific masking of sequences in the left inverted repeat by a ribosome translating the leader. As the A site of a translating ribosome passes leader codon 6, the secondary structure becomes destabilized and a transient opportunity exists for unimpeded entry of a ribosome at RBS-C. However, as the leader ribosome passes to leader codon 7 it is now in a position to compete with nbosome entry at RBS-C. A loop insertion mutation of 3 nt, for example, is thought to prevent the ribosome passing leader codon 7 from competing with the transient opportunity of a ribosome to initiate at RBS-C.

The above scheme for ribosome destabilization of the cat secondary structure differs from that suggested as operating in transcription attenuation {Landick and Yanofsky, 1987). In the latter, a stalled ribosome non-speoifically masks sequences in adjacent secondary structure which causes the RNA to fold in an alternative conformation, thereby relieving a downstream transcription pause or termination function. In contrast, in the cat system, ribosome destabilization of the secondary structure is not predicted to provoke an alternative folding arrangement within the mRNA. Rather, the ribosome must destabilize the secondary structure and maintain the destabilized state to permit repeated ribosome entry at RBS-C.

Our results imply that a drug-stalled ribosome in the leader is essentially adjacent to a ribosome initiating translation at RBS-C. However, this implication is probably incorrect. For example, 50 nt separate identical sites on a drug-stalled ribosome and an initiating ribosome during cat-86 induction; the A site of the stalled ribosome occupies leader codon 6 and the A site of the initiating ribosome is predicted to occupy caf codon 2. Fifty nucleotides is unlikely to represent the maximum packing density of ribosomes on mRNA. It has been shown, for example, that 27 nt is the centre-to-centre distance between eukaryotic ribosomes that are caused to pile up by preventing translation elongation (Wolin and Walter, 1988). We can suggest two reasons why the 50 nt spacing may be appropriate in cat regulation. First, the spatial relationship between crb and the left inverted repeat is critical for induction. We feel this indicates that the stalled ribosome contacts, in a highly precise manner, both crb and specific sequences in the left inverted repeat (e.g. anti-RBS-C). Such a proposed mechanism for melting the secondary structure may shorten the actual distance between the stalled and initiating ribosomes. Second, spatial relationships deduced from the proximity between ribosomes during pile up, or from the number of mRNA nts protected from ribonuclease or dimethylsulphate by ribosome binding, represent the minimum domain of RNA contacted by a ribosome. Evidence from studies with Escherichia co//suggests that a much larger region of RNA may be required for efficient translation initiation (Gold and Stormo, 1987).

The tight control of the site of drug-dependent ribosome stalling by leader and secondary structure sequences predicts that the site of entry of the cat initiating ribosome, at RBS-C, must be a sufficient distance downstream to prevent competition between the two ribosomes. Therefore It seemed logical to conclude that a second spatial relationship of high significance for cat regulation would involve the distance between the stalled ribosome and RBS-C. To measure this distance we have used the spacing between crb in the leader and RBS-C. In wild-type cat-86 this interval is 33 nt and in cat-194 the same intervals is 36 nt; the interval in other inducible caf genes falls between these extremes. Reducing this interval in cat-86 by 3 or 6 nt, through deletions within the loop, diminished induced expression by about 50 and 85%, respectively, and a 9 nt deletion from the loop eliminated induction. These decreases in inducible expression are probably caused by placing into competition the initiating ribosome with the stalled, regulating ribosome. The supposition that in wild-type cat-86 the stalled and initiating ribosomes do not compete is supported by the observation that increasing the distance between crb and

The precise spatial relationships that govern cat-86 regulation are conserved among several caf genes (Fig. 1), and seem necessary if the caf regulatory unit is to function in the manner proposed for translation attenuation based on a minimum of sequence information. The brevity of the cat regulatory sequences become clearly evident when


Z. Gu and P. S. Lovett


Insertion/ Deletion


- 9

- 24.6

- 6

Fig. 4. Computer-predicted folding ot the mRNA secondary structure resulting trom insertion and deletions in the loop. 'Zero' insertion/deletion is the secondary structure that negatively regulates wild-type oat-86. The computer program used was based on the algorithm of Zuker and Stiegler (1981).

,T-A-G-A-G-G-A-G-G-A-C-T-T-a X-ao Pt C-A


- 24.3


V T T ^ ; T ^ ; T ? ? /




- 24.1 _|

3 01--



+ 3






G-G-A-C-T-T-f=l T-8-A-A ,T-R-6-A-G-G-A-G-G-A-C-T-T-^ -rl 50

- 30.3

+ 10

- 28.6

+ 13


Cspft 30


^t-A-G-A-G-G-A-G-G-A-C-t-f-A t-'^-i^-


a compared with the larger domains that regulate erm genes (e.g. see Dubnau (1984)). Erythromycin-inducible expression is seen with cat-$6 (Rogers and Lovett, 1990) and therefore the apparently complex regulatory elements associated with erm genes are not essential for an erythromycin-induction phenotype. We suspect that the minimal nature of the cat regulatory domain may require a greater participation of the regulating ribosome than is necessary for erm induction.

host. Plasmid pPL703 and mutant derivatives were used throughout. pPL703 consists of the replication origin and the neomycinresistance gene of pUBIIO joined to the promoterless cat-86 gene (Mongkolsuk ef ai, 1983). In all constructions cat-86 was activated with the P4 promoter (Mongkolsuk etai, 1983). Growth conditions and methods for plasmid isolation and transformation were as described previously (Lovett and Keggins, 1979).

Assay for chloramphenicol acetyltransterase (CAT)

CAT was assayed by the colorimetric method of Shaw (1975). Assays were performed at 25°C. Protein was measured by the Experimental procedures method of Bradford (1976). CAT-specific activity is reported as p.moles of chloramphenicol acetylated per min per mg ot protein. Inductions were performed by adding 2 tvg m l " ' of chlorampheniBacteria, plasmids and growth conditions col to log-phase cells (38-40 Klett Units) grown with shaking in Bacittus subtitis strain BR151 (trpC2, metB 10, lys-3) was piasmid penassay broth. Incubation was continued for 2h at 37°C.

cat gene regutation 2775 Cell-free extracts were prepared and assayed for CAT. Each CAT value reported is the average of at least four separate determinations, which varied by no more than ±10%.

Site-directed mutagenesis Mutagenesis was performed using the method of Taylor ef ai (1985) as previously described (Duvall ef ai, 1987) with M13 vectors (Zoller and Smith, 1983). Sequencing was by the dideoxy method (Sanger et ai, 1977). Mutations made in Ml3 vectors were transferred to pPL703 by cloning, and the presence of the mutation in pPL703 was confirmed by sequencing directly from the plasmid. A series of insertions and deletions in the loop region of the cat-86 regulatory domain was constructed (Fig. 4). The free energies of the resulting secondary structures were determined by the algorithm of Zuker and Stiegler (1981). The highest free energy was obtained with the - 9 loop deletion; this value of -23.2 is below the free energy ofthe caf-194 secondary structure (AG - -21.0). The rationale for making alterations in the sequence of the loop was based on previous studies which indicated that the sequence of nucleotides in the loop was probably unimportant in terms of induction (see Duvall ef ai, 1987). Furthermore, the creation of a restriction site in the loop or inserting translation stop codons into the loop while maintaining loop size was without effect on basal or induced caf expression (Laredo efa/., 1988; Rogers efa/., 1991).

Acknowledgements We thank Elizabeth Rogers, Nick Ambuios, Phil Farabaugh and Oscar Miller for discussion, information and comments on the manuscript. This investigation was supported by grants from the National Institutes of Health (GM-42925) and the National Science Foundation (DMB-8802124).

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Dubnau, D. (1984) Translational attenuation: the regulation of bacterial resistance to the macrolide-lincosamide-streptogramin B antibiotics. Crit Rev Biochem ^6:103-132. Duvall, E.J., and Lovett, P.S. (1986) Chloramphenicol induces translation of the mRNA for a chloramphenicol resistance gene in Baciltus subtitis. Proc Natt Acad Sci USA 83: 3939-3943. Duvall. E.J.. Williams, D.M., Lovett, P,S., Rudolph. C , Vasantha, N., and Guyer, M. (1983) Chloramphenicol inducible gene expression in Bacitlus subtitis. Gene 24:171-177. Duvall, E.J., Williams, D.M., Mongkolsuk, S., and Lovett, P.S, (1984) Regulatory regions that control expression of two chloramphenicol-inducible caf genes cloned in Bacillus subtitis. J Bacteriot 158: 784-790. Duvall. E.J., Mongkolsuk, S., Kim, U.J., Lovett, P.S., Henkin, T.M., and Chambliss. G,H. (1985) Induction of the chloramphenicol acetyltransferase gene caf-86 through the action of the ribosomal antibiotic amicetin: involvement of a Bacitlus subtilis ribosomal component in caf induction. J Bacteriot 161: 665672. Duvall, E.J., Ambuios, Jr, N.P,. and Lovett. P.S. (1987) Drug-free induction of a chloramphenicol acetyltransferase gene in Bacittus subtitis by stailing ribosomes in a regulatory leader. J Bacteriot 189: 4235-^241. Gold. L., and Stormo, G. (1987) Translational Initiation. In Escherichia coli and Salmonella typhimurium; Cettutar and Motecutar Biotogy. Neidhardt, F.C, Ingraham, J.L., Low. K.B.. Magasanik, B., Schaechfer, M., and Umbarger, H.E. (eds). Washington, D.C: American Society for Microbiology, pp. 1302-1307. Gold, L., Pribnow. D.. Schneider, T., Shinedling. S., Singer, B.S,. and Stormo, G. (1981) Translation initiation in prokaryotes. Annu Rev Microbiot 35: 365-403. Green, CJ., Stewart, G.C, Holtis. M.A., Void, B.S., and Bott, K.F. (1985) Nucleotide sequence of the Bacittus subtitis ribosomal RNA operon, rrnB. Gene 37: 261-266. Horinouchi. H., and Weisblum. B. (1982) Nucleotide sequence and functional map of pC194. a plasmid that specifies inducible chloramphenicol resistance. JSacfeno/150: 815-825. Landick, R.,and Yanofsky, C (1987) Transcription attenuation. In Escherichia coli and Salmonella typhimurium.- Cellular and Motecutar Biotogy. Neidhardt, F.C. Ingraham, J.L., Low, K.B.. Magasanik, B.. Schaechfer. M., and Umbarger. H.E. (eds). Washington, D.C: American Society for Microbiology, pp. 1276-1301. Laredo. J., Wolff, V., and Lovett, P.S. (1988) Chloramphenicol acetyltransferase specified by cat-86: gene and protein relationships. Gene 73: 209-214. Lovett, P.S. (1990) Translational attenuation as the regulator of inducible caf genes. J Bacteriot 172:1-6. Lovett, P.S., and Keggins, K.M. (1979) S. subtilis as a host for molecular cloning. Meth EnzymotBQ: 342-357. Mongkolsuk, S.. Chiang. Y.-W.. Reynolds. R.B.. and Lovett, P.S. (1983) Restriction fragments that exert promoter activity during postexponential growth of Bacittus subtitis. J Bacteriot 155: 1399-1406. Rogers. E.J.. and Lovett, P.S. (1990) Erythromycin induces expression of the chloramphenicol acetyltransferase gene cat-86. J Bacteriot 172: 4694-4695. Rogers, E.J., Ambuios, Jr, N.P., and Lovett, P.S. (1990a) Complementarity of Bacillus subtitis 16S rRNA with sites of antibioticdependent ribosome stalling In caf and erm leaders. J Bacteriol 172:6282-6290. Rogers. E.J.. Kim, U.J., Ambuios, Jr, N.P.. and Lovett. P.S. (1990b) Four codons in the cat-86 leader define a chloramphenicol-sensitive ribosome stall sequence. J Sacfeno/172:110115.


Z Gu and P. S. Lovett

Rogers, E.J., Ambulos, Jr, NN.P., and Lovett, P.S. (1991) Ribosome hopping and translational frameshifting are inadequate alternatives to translational attenuation in cat-86 regulation. J Bacteriol 173: 7881-7886. Sanger, F., Nicklen, S., and Coulson, A.R- (1977) DNA sequencing with chain-terminating inhibitors. Proc NatI Acad Sci USA 74: 5463-5467. Shaw, W.V. (1975) Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Meth Enzymol A3: 737-755. Shaw, W.V., Brenner, D.G., LeGrice, S.F.J., Skinner, S.E., and Hawkins, A.R. (1985) Chloramphenicol acetyltransferase gene of staphylococcal plasmid pC22^. FEBS Lett 179:101-106. Taylor, J.W., Ott, J., and Eckstein, F. (1985) The generation of oligonucleotide directed mutations at high frequency using phosphorothioate-modified DNA. NucI Acids Res 13: 87658785.

Weisblum, B. (1983) Inducible resistance to macrolides, lincosamides and streptogramin B type antibiotics: the resistance phenotype. its biological diversity and structural elements that regulate expression. In Gene Function in Prokaryotes. Beckwith, J., Davies, J., and Gallant, J.A. (eds). Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, pp. 91-121. Wolin, S.L., and Walter, P. (1988) Ribosome pausing and stacking during translation of a eukaryotic mRNA. EMBO J 7: 35593569. Zoller, M.J., and Smith, M. (1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned into Ml 3 vectors. Meth Enzymol 100: 485-500. Zuker, M., and Stiegler. P. (1981) Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. NucI Acids Res 9:133-144.

Perturbing highly conserved spatial relationships in the regulatory domain that controls inducible cat translation.

Chloramphenicol activates translation of cat-86 mRNA by stalling a ribosome in the leader of individual transcripts. Stalling triggers two sequential ...
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