Mistranslation during phenylalanine starvation

Mol Gen Genet (1986) 204:70-74 MGG © Springer-Verlag 1986

Mistranslation during phenylalanine starvation Jack Parker and James Precup Department of Microbiology, Southern Illinois University, Carbondale, IL 62901, USA

Summary. Starvation for phenylalanine led to leucine misincorporation frequencies of 0.1 and 0.6 at UUC codons in the argI transcript of Escherichia coli, but no detectable misincorporation at a U U U codon. Under similar starvation conditions the relative synthesis of full sized MS2 coat protein, encoded by the RNA virus or a DNA copy, is greatly reduced, preventing analysis of the protein. This reduction in amount is unaffected by a rpsL mutation.

Key words: Codon misreading - Codon usage - Translation

Introduction

The early elucidation of the genetic code was accompanied by information on the most likely errors which would occur during translation. The first codon to be deciphered, the phenylalanine codon UUU, was found to be misread prima- rily as leucine at a frequency which was dependent on the experimental conditions used (see Nirenberg and Jones 1963; Weinstein et al. 1966; Woese 1967). In vitro protein synthesis, often using poly U, continues to be used to study the mechanism and accuracy of protein synthesis. A poly U dependent system has been developed in which the substitution of leucine for phenylalanine can be kept to frequencies as low as 10-* (Jelenc and Kurland 1979). This error level agrees closely with some early in vivo measurements of amino acid replacement frequencies (Loftfield 1963; Loftfield and Vanderjagt 1972; Edelmann and Gallant 1977), but the exact nature of some of the latter were not unambiguously determined and none involved phenylalanine codons.

Very little information is available on the type, frequency or consequences of codon misreading in vivo. No information is available on missense misreading of U U U codons. It is clear that the basaMevel misreading of certain sense codons can equal or exceed 5 x 10 3 (Bouadloun et al. 1983; Parker et al. 1983), but misreading at other codons must be much lower (Ellis and Gallant 1982; Parker et al. 1983) and context effects have been noted (Johnston et al. 1984). Read-through of stop codons is very context dependent (Bossi and Roth 1980; Bossi 1983; Miller and Albertini 1983), and can occur at frequencies of as high as 0.1 (Kha- zaie et al. 1984). Read-through of stop codons is actually required for the synthesis of certain viral proteins (see Yarus

Offprint requests to: J. Parker

and Thompson 1983). Context dependent ribosomal frameshift errors are also known to occur in vivo, and some of these are at or near phenylalanine codons (Atkins et al. 1983; Dunn and Studier 1983; Fox and Weiss-Brummer 1980). In Escherichia coli the synthesis of release factor 2 may require a frameshift "e r ro r" (Craigen et al. 1985).

The difficulty in studying such errors in vivo is that they are not heritable and apparently many of them occur at relatively low frequencies. In in vitro systems the misreading frequency can be inceased considerably by depriva- tion of the cognate aminoacyl-tRNA (see Nirenberg and Jones 1963). Starvation for particular amino acids has also been found to increase the in vivo error level in relA mutants of E. coli and in certain mammalian cell lines (O'Far- rell 1978; Parker et al. 1978). By sequencing mistranslated protein we demonstrated that the increase in codon misreading during amino acid starvation is a result of a propor- tional increase in the error level at each affected codon (Johnston et al. 1984). Therefore the technique can be used to examine differences in misreading frequencies between codons. We have demonstrated that asparagine starvation increases the misreading at the asparagine codons approximately 100-fold (Johnston et al. 1984). This increase is easily quantitated because the replacement amino acid is lysine and therefore the mistranslated and native species differ in charge and can be separated by isoelectric focusing. If the replacement amino acid for phenylalanine is leucine there would be no charge change and the mistranslated and native species would electrophorese as a mixture. How- ever, if the misreading frequency is as high as that occuring during asparagine starvation (0.1-0.6), such errors should be detectable by sequencing. In this paper we describe the results of experiments in which we used phenylalanine starvation to induce errors in vivo.

Materials and methods

Bacteria and plasmids. All bacteria used were derivatives of Escherichia coli K-12 and are given in Table 1. The plasmid pAil01 (Bencini et al. 1983) was supplied to us by D. Bencini and pPLaACR26 (Remaut et al. 1982) by W. Fiers. The plasmid pSIU510 was constructed as described in Results.

DNA manipulations and sequencing. Plasmids and DNA fragments were purified as described (Maniatis et al. 1982). Restriction endonucleases and ligase were used as per the

71

Table 1. List of strains

Strain Genotype

JK460 JK464 JK481 JK482 KA197

F + , lacl Q, pheA, reIA Hfr, pro-lac, argR, pheA, relA, thi, )~- rpsL derivative of JK460 rpsL derivative of JK464 Hfr, pheA, relA, spoT, ~,-

directions of the suppliers (Bethesda Research Laborato- ries, Gaithersberg, MD and New England Biolabs, Beverly, MA). The sequences of the genes encoding the amino termi- nal region of the proteins were confirmed using the dideoxy chain terminator method following the protocols of Barnes et al. (1983).

Media, growth, starvation, and labeling. The minimal medium used was buffered with 4-morpholine propane sulfo- nic acid (Neidhardt et al. 1974) and supplemented with glu- cose (0.4% w/v), thiamine (5 pg/ml), and required amino- acids (50 gg/ml). Strains containing pSIU510 were grown in 10 gg/ml tetracycline. Strains containing pAil01 were also supplemented with tetracycline and 20 gg/ml uracil in addition to requirements.

Cultures were grown in flasks at 37 °C with rotary shak- ing. Growth was monitored by absorbance at 420 nm. Star- vation was routinely accomplished by collecting cells on a filter, washing with prewarmed medium without the required amino acid and resuspending the cells in the same (Parker et al. 1980). Infection with MS2 was also accomplished as previously described (Parker et al. 1980). For experiments involving production of coat protein from the plasmid pSIU510, the inducer isopropylthiogalactoside (IPTG) was added to J mM.

Cells were labeled by the addition of [asS]-L-methionine to 10 gCi/ml (ca. 1 Ci/mmol) or [3H]-L-leucine to 5 gCi/ml (5 Ci/mmol). Labeling times are given in Results.

Electrophoresis, protein purification and sequencing. Coat protein was separated either by the two dimensional none- quilibrium electrophoresis method of O'Farrell et al. (1977) or on one dimensional sodium dodecyl sulfate gels (Laemmli 1970). Ornithine transcarbamylase was separated using the two dimensional, equilibrium pH gradient method of O'Farrell (1975).

Pure protein was isolated directly from the polyacryl- amide gels and subjected to automated Edman degradation as described (Johnston et al. 1984). The N-butyl chloride fractions from the sequencer were dried and radioactivity was determined using a scintillation counter.

Results

Phenylalanine starvation and the synthesis of MS2 coat protein

MS2 coat protein has two phenylalanine residues near its amino terminus: that at position 4 encoded by a U U U and that at position 7 encoded by UUC. We reasoned that a substitution by leucine at either would be easily detectable in a mixture if it occured at a frequency of 0.02 or above.

A culture of KAI97 was infected with the RNA virus MS2, treated with rifampicin to stop host RNA synthesis,

c~ m . o a ~ t n r~

H H H ~ H M H H M

0 0 ~ ~ ~

Imbd I I I I I ti 1 ir I I I I | ,

0 I l I I 2 3 I 4 Ptac I ~ I

I

(pBR322)

ori Tet r

H

ua

I 7 . 4 / 0

. . . .

Reading 1 ~ F r a m e - 1 I r / / / / / / / / / / / / / / / / / / A I [ , I / / 3 I

I M a t u r a s e I Lysis | I

Reading I Coat R e p l i c a s e

I V / / / . , ~ / A V / / / / / / / / / / / / / / / / / / / / / / / / / 4 F r a m e 0 I

Fig. l . Restriction map of pSIU510. A linearized representation of the plasmid is shown at the top of the figure with distances in kilobases. The tac promoter is shown as the thick black arrow and the pBR322 port ion as a thick black line. Numbers in parenthe- ses above the restriction sites in the MS2 portion refer to the base pair which corresponds to the nucleotide from the 5' end of the MS2 genome. Below the map the MS2 port ion is expanded to show the location of the reading frames of the four MS2 genes

and then starved for phenylalanine. The residual rate of protein synthesis fell only to 18%. The cells were labeled with [3H]-leucine, coat protein was isolated by two-dimensional electrophoresis and subjected to automated Edman degradation. The counts at position 4 were slightly above background and could have represented a mistranslation frequency of approximately 0.0J (data not shown). Howe- ver, similar results were obtained with unstarved cells (T.C. Johnston and J. Parker, unpublished results). If a high basal-level error occurs at this U U U codon, we reasoned that starvation should have increased it. In order to make the starvation more severe we repeated the experiment but omitted the rifampicin so that the continued synthesis of host protein would further drain the phenylalanine pool. Under these conditions the residual rate of protein synthesis fell to 5%, but unexpectedly the relative rate of coat protein synthesis was also much lower (data not shown, see below).

To further investigate this phenomenon we constructed a plasmid which had a cDNA copy of the coat protein gene under the control of an inducible promoter. The plasmid pPLaACR26 was digested with the restriction endonucleases EcoRI (partial) and PstI (to completion) and the fragment containing the duplex cDNA copy of MS2 purified. This fragment was ligated into the plasmid pBR322 from which the small EcoRI-PstI fragment had been remo- ved. Included in the ligation mixture was a DNA fragment containing the tacI promoter with EcoRI complementary ends (de Boer et al. 1983). The resultant plasmid, pSIU510 (see Fig. 1), confered tetracycline resistance and carried the MS2 genome under the control of the tac promoter. The plasmid could be stably maintained in strains carrying the lacl Q mutation on the chromosome. When induced with IPTG such a strain synthesizes coat protein as approximately 15% of total protein at 3~40 minutes post-induction, (at about 50 minutes it lyses because of the production of lysis protein).

A culture of JK460/pSIU510 was grown, induced by the addition of [PTG and 20 minutes later a portion of the culture was starved for phenylalanine and another for isoleucine (by valine addition to 200 pg/ml). These cells

72

Fig. 2. Autoradiogram of proteins synthesised in induced cultures containing the plasmid pSlU510, starved and unstarved. Strain JK460 containing pSIU510 was grown, induced with IPTG, and labeled for t0 min with [35S]-L-methionine as described in Mate- rials and methods. Total protein was separated on SDS polyacryl- amide gels. The arrows point to coat protein. Lane A is from induced cells labeled beginning 15 rain after phenylalanine starvation; lane B from unstarved cells, and lane C from cells labeled beginning 15 rain after valine addition

were then labeled with [35S]-methionine, extracts were made and a gel run. Fig. 2 shows an autoradiogram of the gel. It is quite clear that phenylalanine starved cells (lane A) make much less coat protein than do cells starved for isoleucine (lane C), exactly the same as found in the experiment with infected cells. Repeating the experiment with a host strain (JK481) which contained the strA1 (rpsL) alMe gave essentially identical results (data not shown). Therefore if an error on the ribosome is responsible for the decrease in coat protein, this error is not significantly reduced by streptomycin resistant ribosomes. Since coat protein seemed to be preferentially affected by this phenomenon we decided to assay phenylalanine codon misreading using a different gene.

Ornithine transearbamylase synthesized during phenylalanine starvation

The argI gene of E. coli, which encodes one of the ornithine transcarbamylase isozymes, has been sequenced and cloned onto a high-copy number plasmid under the control of its own promoter (Bencini et al. 1983). The argI gene encodes 12 phenylalanine residues: 7 by UUC and 5 by UUU. Three phenylalanine residues are near the amino-terminus of the protein, at position 3 (UUU), 8 (UUC) and 14 (UUC). In addition there are leucine residues at positions 9, 11, and 12 which can be used for quantitation of repetitive yield during protein sequencing.

The plasmid, pAil01, is maintained in strain JK464. This strain is deleted for argF but not argI and also contains a mutation in argR, the gene encoding the arginine regulon repressor protein. The presence of the plasmid in the strain increases the mass doubling time of the culture in minimal medium from 70 to 110 minutes. Figure 3 shows autoradio-

Fig. 3A, B. Autoradiograms of two-dimensional gels of proteins synthesised in cultures of JK464 with and without the plasmid pAil01. Cells were grown and labeled in mid-log phase with [35SJ- L-methionine as described in Materials and methods. Both gels are oriented with the most acidic proteins on the right. The arrows point to elongation factors G (G) and Tu (Tu), and to ornithine transcarbamylase (OTC). A is from the strain containing no plasmid and B from the same strain but containing the plasmid pAil01

grams of two dimensional gels prepared with extracts of [35S]-methionine labeled cultures of JK464 with (panel B) and without (panel A) the plasmid. In the plasmid containing strain, ornithine transcarbamylase is synthesized con- stitutively and accounts for over 30% of the label. Starva- tion for phenylalanine reduces this to approximately 15% between 30 and 60 rain after starvation. The enzyme labeled with [3H]-leucine in control and starved cultures was purified and sequenced. Figure 4 shows the radioactivity recove- red from each sequencing cycle. The protein made in unstarved cells (open circles) shows counts only at residues encoded by leucine codons. However, the protein made in phenylalanine starved cells (closed circles) also contained radioactivity at positions 8 and 14. Codon usage was confirmed by sequencing the DNA from this region of the plasmid before and after the experiments (results not shown).

The data from three separate experiments was essentially identical (although in one case the sequencing was not carried out to residue 14 in the starved sample). In no case were counts significantly above background detec- ted at position 3. The counts at positions 8 and 14 indicated a substitution frequency of leucine for phenylalanine of 0.6

73

3600

3200

" E 2400

"~' 2000 U

i 1600

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

C Y C L E N U M B E R

Fig. 4. Sequence analysis of ornithine transcarbamylase from starved and unstarved cells. Strain JK464/pAI101 was labeled with [3H]-L-leucine as described in Materials and methods. Ornithine transcarbamylase was purified on two dimensional gels, eluted and subjected to automated Edman degradation. The open circles are the counts in each cycle from unstarved cells; the closed circles are counts from cells labeled from 30-60 rain after phenylalanine starvation

and 0.1, respectively. In a separate experiment using JK482, a rpsL derivative of JK464, no leucine above background was found incorpora ted at phenylalanine codons (data not shown).

Discussion

Starvat ion for phenylalanine led to a frequency of leucine mis incorpora t ion of 0.6 at one U U C codon, 0.1 at another and no measurable mis incorpora t ion at a U U U codon in the argI transcript . The high level of mistranslat ion found at the U U C codons is compat ible with that found at an A A U codon during previous studies (Johnston et al. 1984). The difference in substi tut ion frequencies at U U C codons indicates strong context effects, a l though some component of differential turnover has not been ruled out. The difference between U U U and U U C codons could involve both codon specific and context specific effects. I t can not be that all U U U codons are misread at a high frequency (0.6 or above) under this condi t ion and the mistranslated products are abor ted (e.g. editing; see Caplan and Menninger 1979) or rapidly degraded. I f this were true one would ex- pect very little full length ornithine t ranscarbamylase from a transcript containing 5 U U U codons, and that is not the case. I t could be true, however, that frameshifting is the prefered error at this par t icular codon or that editing is par t icular ly effective here.

The lack of measurable misreading at the U U U codon was unexpected. In E. coli, codons ending with U are avoi- ded in cases where third posi t ion misreading will lead to an amino acid substitution. There is considerable evidence that most A A U codons are mistranslated more frequently

than A A C codons. We hypothesized (Parker et al. 1983; Johnston et al. 1984) that all such N N U codons m a y have high basal level error frequencies and thus be selected against in genes encoding abundan t proteins. The results reported here with U U U might argue against this simple model. These results also argue for extreme caut ion when extrapolat ing da ta from missense errors in in vitro poly U dependent systems to the in vivo situation. However, we have not demonst ra ted conclusively that errors do not occur at this codon, only that we did not recover protein containing such errors.

Severe phenylalanine deprivat ion led to a specific relative decrease in the synthesis of the MS2 coat protein wheth- er encoded by viral R N A or a duplex D N A gene under the control of the tac promoter . This phenomenon was not seen during starvat ion for isoleucine (Fig. 2), asparagine, lysine, arginine, or proline (Parker et al. 1980), nor did we observe it with any other protein. Therefore we feel that this is not simply premature terminat ion at stalled r iboso- mes, nor is it the result of general editing or turnover func- tions. However, wi thout identif ication of the products it is not possible to establish the precise nature of the event. It seems most probable , however, that it is a context dependent frameshift error; one that is insensitive to the rpsL mutat ion, which has previously been found to suppress many codon misreading errors, including frameshifts (Weiss and Gal lant 1983). Lack of sensitivity to this muta t ion would also be expected for the synthesis of any essential E. coli protein requiring a frameshift error.

These results indicate that our knowledge of the mecha- nisms controll ing the accuracy of protein synthesis is very limited. Indeed we have few measurements on how accurate it actually is. We believe though that it is now clear that there are both codon and codon context effects. Addi t io- nally proteins containing errors are not always efficiently (i.e. preferentially) degraded (Parker 1981) and the results with coat protein might indicate that some misreading can not be " c u r e d " by simply using s t reptomycin resistant mutants. We believe that much remains to be learned about the structure of the coding regions of genes.

Acknowledgements. We thank H. de Boer, Genentech, for the tac promoter, D. Bencini, Harvard, for the plasmid pAil01, and B. Bachmann, Yale, for the strain KA197. The protein sequencing was performed at the Protein Sequencing Laboratory, Washington University, under the supervision of G. Grant. We thank R. Epler Connors for technical assistance and T.C. Johnston and A.K. Ul- rich for advise and helpful discussions. This work was supported by the National Institutes of Health through grant GM25855.

References

Atkins JF, Nichols BP, Thompson S (1983) The nucleotide sequence of the first externally suppresible - 1 frameshift mutant, and of some nearby leaky frameshift mutants. EMBO J 2:1345 1350

Barnes WM, Bevan M, Son PH (1983) Kilo-sequencing: creation of an ordered nest of asymmetric deletions across a large target sequence carried on phage MI 3. Methods Enzymol 101:98-122

Bencini DA, Houghton JE, Hoover TA, Foltermann KF, Wild JR, O'Donovan GA (1983) The DNA sequence of argI from E. coli K-12. Nucleic Acid Res 11:8509-8518

Bossi L (1983) Context effects: translation of UAG codon by sup- pressor tRNA is affected by the sequence following UAG in the message. J Mol Biol 164:73-87

74

Bossi L, Roth JR (1980) The influence of codon context on genetic code translation. Nature 286:123-127

Bouadloun F, Donner D, Kurland CG (1983) Codou-specific missense errors in vivo. EMBO J 2:1351-1356

Caplan AB, Menninger JR (1979) Tests of the ribosomal editing hypothesis: amino acid starvation differentially enhances the dissociation of peptidyl-tRNA from the ribosome. J Mol Biol 134: 621-637

Craigen WJ, Cook RG, Tate WP, Caskey CT (1985) Bacterial peptide chain release factors : Conserved primary structure and possible frameshift regulation of release factor 2. Proc Natl Acad Sci USA 82: 361 6-3620

de Boer HA, Comstock LJ, Vasser M (1983) The tae promoter: a functional hybrid derived from the trp and lae promoters. Proc Natl Acad Sci USA 80:21-25

Dunn JJ, Studier FW (1983) Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 Genetic elements. J Mol Biol 166:477-535

Edelmann P, Gallant J (1977) Mistranslation in E. coli. Cell 10:131-137

Ellis N, Gallant J (1982) An estimate of the global error frequency in translation. Mol Gen Genet 188:169-172

Fox DF, Weiss-Brummer B (1980) Leaky + 1 and - 1 frameshift mutations at the same site in a yeast mitochondrial gene. Nature 288 : 60-63

Jelenc PC, Kurland CG (1979) Nucleoside triphosphate regenera- tion decreases the frequency of translation errors. Proc Natl Acad Sci USA 76:3174-3178

Johnston TC, Borgia PT, Parker J (1984) Codon specificity of starvation induced misreading. Mol Gen Genet 195 : 459-465

Khazaie K, Buchanan JH, Rosenberger RF (1984) The accuracy of Q[3 RNA translation 1. Errors during the synthesis of Q[3 proteins by intact Escherichia coli cells. Eur J Biochem 144:485-489

Laemmli UK (1970) Cleavage of the structural proteins during the assembly of the bacteriophage T4. Nature 227:68(~685

Loftfield RB (1963) The frequency of errors in protein biosynthesis. Biochem J 89 : 82-92

Loftfield RB, Vanderjagt D (1972) The frequency of errors in protein biosynthesis. Biochem J 128:1353-1356

Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, New York

Miller JH, Albertini AM (1983) Effects of surrounding sequence on the suppression of nonsense codons. J Mol Biol 164:59-71

Neidhardt FC, Bloch PL, Smith DF (1974) Culture medium for enterobacteria. J Bacteriol 119 : 736-747

Nirenberg MW, Jones OW, Jr (1963) The current status of the RNA code. In: Vogel H J, Byrson V, Lampen JO (eds) Informa- tional Macromolecules. Academic Press, New York, pp 451465

O'Farrell PH (1975) High resolution two-dimensional electrophoresis of protein. J Biol Chem 250:4007-4021

O'Farrell PH (1978) The suppression of defective translation by ppGpp and its role in the stringent response. Cell 14:545-557

O'Farrell PZ, Goodman HM, O'Farrell PH (1977) High resolution two dimensional electrophoresis of basic as well as acidic proteins. Cell 12:1133-1142

Parker J (1981) Mistranslated protein in Escherichia eoli. J Biol Chem 256:9770-9773

Parker J, Pollard JW, Friesen JD, Stanners CP (1978) Stuttering: high-level mistranslation in animal and bacterial cells. Proc Natl Acad Sci USA 75:1091-1095

Parker J, Johnston TC, Borgia PT (1980) Mistranslation in cells infected with the bacteriophage MS2: direct evidence of Lys for Asn substitution. Mol Gen Genet 180:275-281

Parker J, Johnston TC, Borgia PT, Holtz G, Remaut E, Fiefs W (1983) Codon usage and mistranslation: in vivo basal level misreading of the MS2 coat protein message. J Biol Chem 258:10007-10012

Remaut E, De Waele P, Marmenout A, Stanssens P, Fiers W (1982) Functional expression of individual plasmid-coded RNA bacteriophage MS2 genes. EMBO J 1 : 205-209

Weinstein IB, Friedman SM, Ochoa M, Jr (1966) Fidelity during translation of the genetic code. Cold Spring Harbor Symp Quant Biol 31:671-681

Weiss R, Gallant J (1983) Mechanism of ribosome frameshifting during translation of the genetic code. Nature 302 : 389-393

Woese CR (1967) The present status of the genetic code. Prog Nucleic Acid Res Mol Biol 7:107-172

Yarus M, Thompson RC (1983) Precision of protein biosynthesis. In: Beckwith J, Davies J, Gallant JA (eds). Gene function in prokaryotes. Cold Spring Harbor Laboratory, New York, pp 23-63

Communicated by A. B6ck

Received February 19, 1986

Mistranslation during phenylalanine starvation

Documents

Transcript of Mistranslation during phenylalanine starvation