JOURNAL OF BACTERIOLOGY, Aug. 1991, p. 4904-4907 0021-9193/91/154904-04$02.00/0

Vol. 173, No. 15

Copyright © 1991, American Society for Microbiology

Role of Translation of the pheA Leader Peptide Coding Region in Attenuation Regulation of the Escherichia coli pheA Gene NARASAIAH GAVINI* AND LAKSHMIDEVI PULAKAT Russell Grimwade School of Biochemistry, University of Melbourne, Parkville, Victoria 3052, Australia, and Department of Molecular Biology and Biochemistry, School of Biological Sciences, University of California at Irvine, Irvine, California 92717* Received 1 March 1991/Accepted 27 May 1991

In Escherichia coli, the expression of the pheA gene is regulated by attenuation of transcription. To study the molecular details of pheA attenuation, we introduced mutations in the pheA leader peptide coding region and analyzed their effects by using pheA promoter-lacZ gene transcription fusions (pheAp-lacZ). Mutations in the ribosome-binding site (pheAel213) or in the translation initiation codon (pheAe24) of the pheA leader peptide coding region resulted in superattenuation of pheA expression. However, the presence of a stop codon upstream to the tandem phenylalanine codons (pheAe3334) led to an increase in the basal-level expression of pheA. This increase was further enhanced in the presence of prfA release factor mutant. The level of pheA expression in all three mutants was the same when cells were starved for phenylalanine. These results demonstrate that efficient translation of the pheA leader peptide coding region and the position of the ribosome on the leader transcript play decisive roles in the attenuation regulation of pheA.

The Escherichia coli gene pheA encodes a bifunctional enzyme, chorismate mutase/prephenate dehydratase (EC 5.4.99.5/4.2.1.51), which catalyzes the first two of the three steps in phenylalanine biosynthesis from chorismate (17). The expression of this gene is regulated by attenuation at a transcription terminator located upstream from the pheA structural gene (24, 27). According to the tenets of attenuation regulation of biosynthetic operons (12, 25), the amount of charged tRNAPhe in the cell should control the attenuation regulation of pheA. Evidence in support of the involvement of tRNAPhC in the attenuation regulation of pheA expression came from the analysis of trans-acting regulatory mutants of pheA (2, 3, 5, 8). Previously, we have shown that pheR, a trans-acting regulatory gene for pheA, encodes a tRNAPhe molecule and regulates pheA expression via attenuation control of transcription (2, 3, 5). Furthermore, we have analyzed the nature of two of the cis-dominant regulatory mutants of pheA isolated previously (10). The promoterattenuator regions of the pheA gene isolated from each of these mutants were characterized by nucleotide sequencing to identify the location of the mutations which caused elevated levels of pheA expression (2, 4). It was found that the mutations were located in the G+C-rich terminator hairpin specified by the pheA attenuator (4). These mutations destabilized the terminator structure and led to increased readthrough into the pheA structural gene (4). Thus, both trans-acting and cis-acting regulatory mutants analyzed to date influence the expression of the pheA gene via attenuation control. In our attempt to understand the molecular details of attenuation of pheA transcription, we have examined the role of translation of the pheA leader peptide coding region in determining the basal-level expression of pheA. Previously, we showed that the release of the translating ribosome from the pheA leader transcript stop codon UGA plays a crucial role in determining the basal-level expression of pheA (6). When a defective release factor 2 (UGA and UAA *

Corresponding author. 4904

specific) was introduced into the cell, the basal-level expression of pheA decreased by about twofold, indicating that slow dissociation of the translating ribosome from the pheA leader transcript stop codon increases the formation of the terminator and attenuates transcription of the pheA gene (6). In this report, we describe mutations that block normal translation of the pheA leader peptide coding region and analyze their effects on attenuation regulation of pheA. The first mutation was generated by changing the guanine residues (Fig. 1) of the putative ribosome-binding site AGGA of the pheA leader transcript to adenine residues (27). This mutation, designated pheAel213, has a string of four adenine residues in place of the conventional ribosome-binding site (7). This should interfere with the binding of the translating ribosome and decrease the synthesis of the pheA leader peptide. The second mutation designated pheAe24, had the translation initiation codon ATG of the pheA leader transcript replaced by the isoleucine codon ATA. Since ATA is not a known translation initiation codon (7, 9), replacing ATG with ATA should block synthesis of the pheA leader peptide. The third mutation, designated pheAe3334, was constructed by converting the proline codon CCG located upstream to the tandem phenylalanine codons (the fifth codon of the pheA leader peptide coding region; Fig. 1) into a stop codon, UAG. This mutation allowed us to examine the effects of introducing a stop codon just upstream to the predicted 1:2 stem-loop structure of the pheA leader transcript on attenuation regulation of pheA. To generate these mutations, a 508-bp Sau3AI fragment carrying the promoter-attenuator region of pheA (4) cloned into the BamHI site of M13tg130 (11, 14) was used. The oligonucleotide-directed mutagenesis was carried out by using the Oligonucleotide Directed In Vitro Mutagenesis System-Version 2 purchased from Amersham. To do this, chemically synthesized oligonucleotide primers corresponding to the region of interest and containing the complementary sequence with the appropriate base change were hybridized to the single-stranded recombinant phage DNA and extended by using DNA polymerase I Klenow fragment and T4 DNA ligase to generate mutant heteroduplexes. During

VOL. 173, 1991

NOTES

A)

AA

(N26) U C G A A C U U U A A U G G

B)

STOP G

U G C-G P C-G A CGG

U

C-G F U

CG.U

U C C UGCUG T C-G A-U U.G F U-A U-A U-A F U C U-A C-G F U-A U-A A-U R C-G G-C C-G F U-A U-A C-G F U-A RBS

K

H

H

I

P

F

AGGAAACAAACATGAAACACAUACCGUU AA

A

UA

Phe*1213

pbeA24

pheS3334

A A

A

4905

C-G G-C G-C A-U G-C G-C G-C U-AA A A A-U

U C

CG C-G

STOp

C-G UG

UU CG

C-G G-C G-C A-U A-U U U MCM UUJAUUJ

1:2

3:4

G-C

U.G C-G p C-G C-G UG F F R F F F TF U G UUCLAI I V1 UItUUUACCOJ UUUUULLAWU

FIG. 1. The predicted mutually exclusive secondary structures of the pheA leader transcript (1:2, 2:3, and 3:4) (27) involved in the attenuation regulation of pheA are shown. The locations of the mutations in the pheA leader transcript which influence attenuation regulation of pheA are marked, and the mutated bases are shown by the arrows. The deduced amino acid sequence of the pheA leader peptide is shown in single-letter code. The nucleotide sequence shown in panel A starts from the predicted ribosome-binding site (RBS) (27) of the pheA leader transcript, which is marked by the overline. The nucleotide sequence shown in panel B starts from the seventh codon (marked 7) of the pheA leader peptide.

this synthesis, dCTP was replaced by dCTPaS so that the newly synthesized strand was phosphorothioate DNA, which cannot be cleaved by the restriction enzyme NciI. The heteroduplex DNA was treated with Ncil to generate nicks in the nonmutant strand, and by using exonuclease III, most of the nonmutant DNA was removed. The double-stranded cloned circular DNA was then resynthesized by DNA polymerase I and T4 DNA ligase to generate homoduplex mutant molecules. E. coli TG1 was transfected with this mutant phage DNA to prepare phage stocks from plaques for mutant analysis. The mutations were confirmed by dideoxy nucleotide sequencing (22). To isolate DNA fragments carrying the wild-type and mutated pheA promoter-attenuator regions, the recombinant phage DNAs were digested with HindIII and, after filling in the ends with DNA polymerase I Klenow fragment, were redigested with EcoRI. These fragments were then cloned into the EcoRI-SmaI site of the transcription fusion vector pRS551 (23). The plasmid pRS551 carries a gene encoding kanamycin resistance and a polycloning site flanked on either side by a P-lactamase gene and a promoterless lacZ operon. Since the promoterless lacZ operon is located downstream from the polycloning sites, a pheApromoter-lacZ operon transcription fusion was generated by the cloning procedure described above. To obtain single copies

of these transcription fusions, E. coli cells carrying the recombinant plasmids were infected with the bacteriophage XRS88 (23). The presence of a 3' truncated P-lactamase gene and a promoterless lacZ operon in this phage allows homologous recombination between the recombinant plasmids and the X phage as described previously (23), resulting in the generation of recombinant XRS88 bacteriophages which carry the kanamycin resistance marker and the pheAp-lacZ operon fusion. An E. coli strain, NG210, which is pheA lac, was constructed by transfering the Lac- phenotype from E. coli P90C(pro lac) (23) to E. coli JP2255 (pheA tyrA thi) (2) by transducing with P1 bacteriophage by using standard methods (15). This strain was then infected with recombinant XRS88 bacteriophages carrying the wild-type and mutated pheAp-lacZ transcription fusions. The colonies which were kanamycin resistant and blue on Luria-Bertani-5 bromo- 4 chloro 3 indolyl - a - D - galactopyranoside agar plates (14, 15) were selected as lysogens. The monolysogens were selected by identifying the colonies which exhibited the lowest level of P-galactosidase activity from each individual infection experiment and were rechecked by using the 'ter test' as described previously (23). The E. coli cells carrying a single copy of the recombinant XRS88 bacteriophages harboring the wild-type and mutated -

-

-

-

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NOTES TABLE 1.

J. BACTERIOL.

P-Galactosidase activity in E. coli strains carrying the wild-type and mutated XpheAp-lacZ fusions

E. coli strain

Relevant resident fusion pheAp-lacZ

Location of mutation

NG211 NG212 NG213 NG214 NG214::RF1 (prfA mutant)

pheApWT-lacZ pheApel213-lacZ pheApe24-lacZ pheApe3334-lacZ pheApe3334-lacZ

Ribosome-binding site Translation initiation codon 5th codon of leader transcript 5th codon of leader transcript

None

Nature of mutationa

None AGGG to AAAA ATG to ATA CCG to UAG

f-Galactosidase MM MMactivityb + Phed

1,270 16 20 340

1,248

75 20 23 312 1,095

The changed bases are indicated in boldface. b Specific activity of P-glactosidase is expressed in units as described by Miller (15). cE. coli cells were grown in minimal medium (MM) (15) supplemented with glucose (0.2% [wt/vol]), 10 ,ug of thiamine per ml, and 1 mM phenylalanine. d Phenylalanine was added to a final concentration of 5 mM. a

pheAp-lacZ transcription fusions were designated NG211 (XpheApeWT-lacZ), NG212(XpheApel213-lacZ), NG213 (XpheApe24-lacZ), and NG214(XpheApe3334-lacZ), respectively. Effects of mutations in the ribosome-binding site and the translation initiation codon of the pheA leader peptide coding region on the attenuation regulation ofpheA. The pheA leader peptide of the pheAp-lacZ transcription fusion present in E. coli NG212(XpheApel213-lacZ) has a string of four adenine residues in place of the wild-type ribosome-binding site, AGGA. As shown in Table 1, when the cells were grown in the presence of phenylalanine, the ,-galactosidase activity of this strain was decreased by about fourfold compared with the P-galactosidase activity of E. coli NG211(XpheApeWT lacZ). When starved for phenylalanine, the ,-galactosidase activity of E. coli NG212(XpheApel213-lacZ) did not change (Table 1). Thus, the presence of a defective ribosomebinding site for the pheA leader peptide coding region increased the formation of the terminator (the 3:4 stem-loop structure) when the cells were grown in the presence or absence of phenylalanine. Therefore, the pheA leader transcript needs an efficient ribosome-binding site to ensure optimal basal-level expression as well as to complete deattenuation of the pheA gene when the cells are starved for phenylalanine. The ,-galactosidase activity of E. coli NG213 (XpheApe24-lacZ) also showed a four- to fivefold decrease relative to the ,-galactosidase activity of NG211(XpheApe WT-lacZ) in the presence of phenylalanine (Table 1). When starved for phenylalanine, the P-galactosidase activity of this strain, like that of E. coli NG212(XpheApel213-lacZ), did not change (Table 1). Hence, an efficient translation initiation codon for the pheA leader peptide is essential for the appropriate attenuation response of the pheA attenuator. Since either a defective ribosome-binding site or an inefficient translation initiation codon would decrease the synthesis of the pheA leader peptide, the above results demonstrate that efficient translation of the pheA leader peptide plays a crucial role in the attenuation regulation of the pheA gene. Effects of the presence of a stop codon at the 5' side of the tandem phenylalanine codons of the pheA leader peptide

coding region. The leader peptide coding region of the pheApe3334-lacZ transcription fusion contains the stop codon UAG as its fifth codon instead of the proline codon CCG present in the wild-type pheA leader transcript. The 3-galactosidase activity of E. coli NG214(XpheApe3334lacZ) was increased by about sixfold compared with that of E. coli NG211(XpheApeWT-lacZ) in the presence of phenylalanine (Table 1). However, when the mutant cells were starved for phenylalanine, the P-galactosidase activity did not change (Table 1), indicating that the ability of the pheA

attenuator to respond to phenylalanine starvation had been lost. It should be noted that the position of the stop codon UAG present in the pheA leader transcript of pheApe3334 lacZ transcription fusion is just upstream to the predicted stem 1 segment of the pheA leader transcript (Fig. 1). Hence, a translating ribosome that has reached this stop codon would interfere with the formation of the 1:2 stem-loop structure, facilitate the formation of the 2:3 stem-loop structure (the antiterminator), and increase the basal-level expression of pheApe3334-lacZ transcription fusion. To further verify the role of the translating ribosome sitting at theUAG stop codon located at the fifth position of the pheA leader transcript of pheApe3334-lacZ transcription fusion in increasing basal-level expression, we introduced a mutant RF1 (UAG specific) (20, 21) into E. coli NG214. To do this, the temperature-sensitive release factor 1 mutation prfA(Ts) was transfered from E. coli UL213[XA1OC prfA(Ts) TnJO] to E. coli NG214 by P1 transduction as described previously (15, 16, 20). Since the prfA(Ts) mutation is linked to the Tetr transposon TnJO, the transfer of this mutation could be easily identified by selecting for Tetr transductants and rechecking them for temperature sensitivity at 42°C (6, 18, 19). The transductant carrying the prfA(Ts) mutation was designated E. coli NG214: :RF1. The ,-galactosidase activity of E. coli NG214::RF1 was about 15-fold higher than the 3-galactosidase activity of E. coli NG211(XpheApeWT-lacZ) (Table 1) when the cells were grown in the presence of phenylalanine. Thus, the ,-galactosidase activity of E. coli NG214:RF1 in the presence of phenylalanine was equal to the 3-galactosidase activity of E. coli NG211 when the cells were starved for phenylalanine. Therefore, the presence of a defective release factor which would slow the dissociation of the translating ribosome from the UAG stop codon prevented the formation of the 1:2 stem-loop structure and facilitated the formation of the antiterminator, leading to complete derepression of the pheA attenuator. Studies on other well-characterized biosynthetic operons controlled by attenuation (1, 12, 13, 26) clearly show that an efficient attenuation response is dependent on the ability to form the appropriate secondary structures in a given physiological situation. Our results confirm that during the attenuation regulation of pheA, both the efficiency of translation of the pheA leader peptide coding region and the position of the translating ribosome on the leader transcript play crucial roles in determining which one of the alternative secondary structures of the pheA attenuator forms. A decrease in the translation initiation due to the presence of either an inefficient ribosome-binding site [E. coli NG212(XpheApel213 lacZ)] or an inefficient translation initiation codon [E. coli NG213(XpheApe24-lacZ)] leads to superattenuation of pheA

NOTES

VOL. 173, 1991

transcription (Table 1). However, the presence of the stop codon in the pheA leader transcript of XpheApe3334-lacZ did not result in superattenuation. Instead, it led to an increase in the basal-level expression of pheA (Table 1), presumably by increasing the formation of the 2:3 stem-loop structure. Thus, fine tuning the phe attenuation response is determined by the efficiency of translation of the pheA leader peptide coding region, the position of the translating ribosome on the leader transcript, and the efficient release of the translating ribosome from the leader transcript stop codon. REFERENCES 1. Chen, J. W., E. Harms, and H. E. Umbarger. 1991. Mutations replacing the leucine codons or altering the length of the amino acid-coding portion of the ilvGMEDA leader region of Escherichia coli. J. Bacteriol. 173:2328-2340. 2. Gavini, N. 1988. Investigations on the regulation of the Escherichia coli gene pheA. Ph.D. dissertation. University of Melbourne, Melbourne, Australia. 3. Gavini, N., and B. E. Davidson. 1990. The pheR gene of Escherichia coli encodes tRNAPhe, not a repressor protein. J. Biol. Chem. 265:21527-21531. 4. Gavini, N., and B. E. Davidson. 1990. pheAo mutants of Escherichia coli have a defective pheA attenuator. J. Biol. Chem. 265:21532-21535. 5. Gavini, N., and B. E. Davidson. 1991. Regulation of pheA expression by the pheR product in Escherichia coli is mediated through attenuation of transcription. J. Biol. Chem. 266:77507753. 6. Gavini, N., and L. Pulakat. 1991. Role of ribosome release in the basal level expression of the Escherichia coli gene pheA. J. Gen. Microbiol. 137:679-684. 7. Gold, L., and G. Stormo. 1987. Translational initiation, p. 1302-1307. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology, vol. 2. American Society for Microbiology, Washington, D.C. 8. Gowrishankar, J., and J. Pittard. 1982. Regulation of phenylalanine biosynthesis in Escherichia coli K-12: control of transcription of the pheA operon. J. Bacteriol. 150:1130-1137. 9. Hershey, J. W. B. 1987. Protein synthesis, p. 613-647. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology, vol. 1. American Society for Microbiology, Washington, D.C. 10. Im, S. W. K., and J. Pittard. 1971. Phenylalanine biosynthesis in Escherichia coli K-12: mutants derepressed for chorismate mutase P-prehenate dehydratase. J. Bacteriol. 106:784-790. 11. Kieny, M. P., R. Lathe, and J. P. Lecocq. 1983. New versatile cloning and sequencing vectors based on bacteriophage M13. Gene 26:91-99. 12. Landick, R., and C. Yanofsky. 1987. Transcription attenuation, p. 1453-1472. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.),

13.

14. 15. 16. 17.

18. 19. 20.

21. 22.

23. 24.

25. 26.

27.

4907

Escherichia coli and Salmonella typhimurium: cellular and molecular biology, vol. 2. American Society for Microbiology, Washington, D.C. Landick, R., C. Yanofsky, K. Choo, and L. Phung. 1990. Replacement of the Escherichia coli trp operon attenuation control codons alters operon expression. J. Mol. Biol. 216:2537. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Miller, J. H., and A. M. Albertini. 1983. Effects of surrounding sequence on the suppression of nonsense codons. J. Mol. Biol. 164:59-71. Pittard, A. J. 1987. Biosynthesis of the aromatic amino acids, p. 368-394. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology, vol. 1. American Society for Microbiology, Washington, D.C. Roesser, J. R., and C. Yanofsky. 1988. Ribosome release modulates basal level expression of the trp operon of Escherichia coli. J. Biol. Chem. 263:14251-14255. Roesser, J. R., Y. Nakamura, and C. Yanofsky. 1989. Regulation of basal level expression of the tryptophan operon of Escherichia coli. J. Biol. Chem. 264:12284-12288. Ryden, S. M., and L. A. Isaksson. 1984. A temperature-sensitive mutant of Escherichia coli that shows enhanced misreading of UAG/A and increased efficiency for some tRNA nonsense suppressors. Mol. Gen. Genet. 193:38-45. Ryden, M., J. Murphy, R. Martin, L. Isaksson, and J. Gallant. 1986. Mapping and complementation studies of the gene for release factor 1. J. Bacteriol. 168:1066-1069. Sanger, F., A. R. Coulson, B. G. Barrell, A. J. H. Smith, and B. A. Roe. 1980. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J. Mol. Biol. 143:161-178. Simons, R. W., F. Houman, and N. Kleckner. 1987. Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene 53:85-96. Yager, T. H., and P. H. Von Hippel. 1987. Transcript elongation and termination in Escherichia coli, p. 1241-1275. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology, vol. 2. American Society for Microbiology, Washington, D.C. Yanofsky, C. 1988. Transcription attenuation. J. Biol. Chem. 263:609-612. Yanofsky, C., R. L. Kelly, and V. Horn. 1984. Repression is relieved before attenuation in the trp operon of Escherichia coli as tryptophan starvation becomes increasingly severe. J. Bacteriol. 158:1018-1024. Zurawski, G., K. Brown, D. Killingly, and C. Yanofsky. 1978. Nucleotide sequence of the leader region of phenylalanine operon of Escherichia coli. Proc. Natl. Acad. Sci. USA 75: 4271-4275.

Role of translation of the pheA leader peptide coding region in attenuation regulation of the Escherichia coli pheA gene.

In Escherichia coli, the expression of the pheA gene is regulated by attenuation of transcription. To study the molecular details of pheA attenuation,...
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