Báo cáo khoa học: Abortive translation caused by peptidyl-tRNA drop-off at NGG codons in the early coding region of mRNA

Abortive translation caused by peptidyl-tRNA drop-off at NGG codons in the early coding region of mRNA Ernesto I. Gonzalez de Valdivia and Leif A. Isaksson

Department of Genetics, Microbiology and Toxicology, Stockholm University, Sweden

Keywords abortive translation; NGG codons; peptidyl- tRNA drop-off; Escherichia coli; early elongation

Correspondence L. A. Isaksson, Department of Genetics, Microbiology and Toxicology, Stockholm University, S-106 91 Stockholm, Sweden Fax: +46 8 15 51 39 Tel: +46 8 16 41 97 E-mail: leif.isaksson@gmt.su.se Website: http://www.gmt.su.se

(Received 29 June 2005, revised 17 August 2005, accepted 19 August 2005)

doi:10.1111/j.1742-4658.2005.04926.x

In Escherichia coli the codons CGG, AGG, UGG or GGG (NGG codons) but not GGN or GNG (where N is non-G) are associated with low expres- sion of a reporter gene, if located at positions +2 to +5. Induction of a lacZ reporter gene with any one of the NGG codons at position +2 to +5 does not influence growth of a normal strain, but growth of a strain with a defective peptidyl-tRNA hydrolase (Pth) enzyme is inhibited. The same codons, if placed at position +7, did not give this effect. Other codons, such as CGU and AGA, at location +2 to +5, did not give any growth inhibition of either the wild-type or the mutant strain. The inhibi- tory effect on the pth mutant strain by NGG codons at location +5 was suppressed by overexpression of the Pth enzyme from a plasmid. However, the overexpression of cognate tRNAs for AGG or GGG did not rescue from the growth inhibition associated with these codons early in the induced model gene. The data suggest that the NGG codons trigger pep- tidyl-tRNA drop-off if located at early coding positions in mRNA, thereby strongly reducing gene expression. This does not happen if these codons are located further down in the mRNA at position +7, or later.

for gene expression, presumably at the translational level.

As an early step during translation initiation in bac- teria the mRNA is anchored to the 30S ribosomal sub- unit by base pairing between a sequence close to the end of the 16S ribosomal RNA and the Shine–Dal- garno (SD) sequence, a few bases upstream of the initi- ation codon in the mRNA. Even though the SD sequence increases initiation efficiency, mRNAs that lack this sequence can be translated [1] albeit at a lower efficiency [2]. In such case the sequence in the downstream region (DR) immediately following the initiation codon has a large influence on gene expres- sion at the translation level [3]. This effect was origin- ally suggested to be the result of an additional anchoring by base pairing between the DR sequence in mRNA and 16S rRNA but this model has been refu- ted experimentally [4,5]. Many rare codons are used within the first 25 codons in Escherichia coli [6]. Clearly, the early coding region in mRNA is important

A closer study of the DR region has revealed that the nature of the +2 codon can affect expression by up to a factor of 20. A number of codons, but in par- ticular G-rich codons, at this position give low gene expression. The codons that follow in the DR have rel- atively similar effects giving normal gene expression with the notable exception of NGG codons (AGG, CGG, UGG and GGG) as these lower gene expression down to 10–20% also if located at positions +3 to +5. The other G-rich codons GGN and GNG (where N is non-G) do not have this effect in this sequence window. At position +7 all G-rich codons, including NGG give normal expression. This is also the case if they are placed at +11 [3,7–10]. The reduced level of gene expression that is seen for the NGG codons in the early coding region is not the result of lowered

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Abbreviations SD, Shine–Dalgarno; DR, downstream region; Pth, peptidyl-tRNA hydrolase; IPTG, isopropyl thio-b-D-galactoside; Ts, temperature sensitive.

E. I. Gonzalez de Valdivia and L. A. Isaksson Abortive translation due to drop-off at NGG codons

secondary structure

formation. mRNA levels or Rather, some abortive event seems likely. One possibil- ity would be shifting of the translational reading frame giving premature termination at some out-of-phase ter- mination codon. However, this explanation does not seem to be valid. Another possibility would be drop- off of peptidyl-tRNA from the translating ribosome during early elongation [11]. In this case the released peptidyl-tRNA would be cleaved by the peptidyl- tRNA hydrolase (Pth) [12], thereby initiating turnover and re-use of the amino acid residues and the tRNA moiety.

complementation is not

One possible reason for the low gene expression caused by the early NGG codons could be abortive translation as a result of peptidyl-tRNA drop-off at these codons [13]. Such released peptidyl-tRNA is nor- mally hydrolysed by a Pth, thus allowing recycling of both the amino acids and the tRNA. A mutant strain MB01 is available with a pth(Ts) (Table 1). In this mutant strain accumulation of peptidyl-tRNA, that cannot be degraded and recycled, leads to inability to grow at high temperature [14]. Introduction of plasmid pVH1 with its pth+ gene gives partial complementa- tion of the temperature sensitivity of MB01 (Table 2). complete, probably This because of the low copy number of pVH1 [20].

Table 1. Bacterial strains and plasmids.

A mutant E. coli strain is available that has a heat sensitive Pth. At high temperature this mutant does not grow because the accumulated peptidyl-tRNA cannot be degraded [13]. This will give shortage of tRNA as a consequence, which is likely to be one of the reasons for the heat-sensitive phenotype of the mutant [14].

Relevant characteristics References

Strains MC1061 [44]

araD139, D(lacI POZYl) 74, galU, galK, rpsL, D(ara A BC-leu)7697, hsdR, mcrB, SmS, D(lacproB), arg E, ara, gyrA, rpoB, thi

Increased heat sensitivity of the pth mutant strain has been used to demonstrate accumulation of pep- tidyl-tRNA in connection with overexpression of short mini-genes [15–18]. Drop-off at a pair of rare AGA AGG codons has been demonstrated in a natural gene and ribosome stalling and drop-off at a pair of argin- ine codons in mini-genes [19].

MG1655, zdh-925::Tn10, pth(Ts) [43] [21] This work

XAC MG1655 Wild-type MB01 Plasmids lacZ pCMS71 [9,10] Derivative of pCM21 with cloning cassette, AmpR

Protein A¢ pHN109 [30]

pEG998 Derivative of pAB93 with an internal 2A¢ Control gene and cloning cassette, AmpR Derivative of pHN109 with Csp45I [10] restriction site

In the present study we show that the NGG codons CGG, AGG, GGG and UGG, when they are posi- tioned in the early coding region downstream of the initiation codon in a highly expressed reporter gene, inhibit growth of a pth thermo-sensitive (Ts) mutant strain. This effect is not seen for other codons or for the NGG codons if placed at a later position (+7). The results suggest that low gene expression associated with NGG in early coding reading in mRNA is the result of peptidyl-tRNA drop-off from the ribosome during translation in E. coli.

Others arg U+ (tRNAArg4), KanR

Results

pUBS250 pArgUW argU+(tRNAArg4), argW+(tRNAArg5) KanR pVH1 pJMM19 pMO22 pth+, KanR lysV+ (tRNALys), KanR glyU+ (tRNAGly1), TetR [34] [40] [20] [20] [39]

Induction of a test gene with an early NGG codon inhibits growth of a pth(Ts) strain

Table 2. Effect of temperature on growth of strain MB01 with a heat sensitive peptidyl-tRNA hydrolase. Strains were streaked on Luria–Bertani agar plates with IPTG (1 mM) (+) or without (–), and incubated at the indicated temperatures.

IPTG (–) IPTG (+)

Strains Plasmid 30 (cid:1)C 37.5 (cid:1)C 43 (cid:1)C 30 (cid:1)C 37.5 (cid:1)C 43 (cid:1)C

MG1655 – – MB01 pVH1 MB01 ++++ ++++ ++++ +++ ++++ ++++ ++++ +++ ++++ ++++ ++++ – + ++++ ++++ ++++ – – ⁄ +

immediately following the Codons at position +2, AUG initiation codon, influence gene expression by up to a factor of 20. Further down at positions +3 to +5 in the DR this codon influence is less pronounced with the notable exception of the NGG codons that lower gene expression to only 10–20% [10]. The other G-rich codons GGN and GNG (where N is non-G), do not give such low gene expression. The low expres- sion associated with NGG is not seen if the codon is located further down in the mRNA coding region at +7 [10].

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E. I. Gonzalez de Valdivia and L. A. Isaksson Abortive translation due to drop-off at NGG codons

CGU and AGA at +2, +3 and +5 did not inhibit growth of the strain even in the presence of IPTG induction. All of these results suggest that the low gene expression associated with NGG codons in the down- stream region following the initiation codon [9,10], is the result of peptidyl-tRNA drop-off that is excessive enough to inhibit growth of a pth(Ts) mutant strain.

Pth enzyme rescues from growth inhibition by +5 NGG codons

To test for excessive drop-off of peptidyl-tRNA at certain early codons (including the NGG codons) they were placed in the 5¢-coding region of a lacZ reporter gene in a plasmid (Fig. 1A) and introduced into the pth(Ts) mutant strain MB01, with its temperature sen- sitive Pth (Table 1). Cultures were pregrown at 30 (cid:1)C, followed by dilution into the same medium with or without isopropyl thio-b-d-galactoside (IPTG; 1 mm) whereafter growth was continued at 37.5 (cid:1)C. As shown in Fig. 1, induction of test genes with NGG codons at positions +2, +3 and +5 inhibits growth of the pth(Ts) strain. This inhibitory effect is not obtained if the NGG codons are located at +7 or if the test gene is not induced by IPTG. It was noted, but not ana- lysed further, that cells with the nondrop-off codons CGU and AGA at position +7 were delayed in growth in the early growth phase. The arginine codons

As described above, it appeared likely that the inhibi- tory growth effect by early NGG codons in the pth(Ts) mutant MB01 was due to accumulation of peptidyl- tRNA, as the result of an abortive drop-off event. MB01 did not grow at 43 (cid:1)C and it showed disturbed the growth at 37.5 (cid:1)C (Table 2). To confirm that

A

B

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Fig. 1 Plasmid constructs are derivatives of pDA3480 [9,10]. The 5¢ end of the transcript is 5¢-AAUUGUGAGCGGAUAACAAUUUCA- CCAGGUAAUAAAUUAAAUAAAAUUUAAAUAUG-3¢ for the gene variants that lack a functional Shine–Dalgarno sequence (SD, underlined) [21]. (A) LacZ reporter gene construct pCMS71 was used as a cloning vector for the insertion of different AUG downstream context sequences using the restriction sites SwaI ⁄ SalI and SwaI ⁄ Csp45I. The lacZ gene is under control of the trc promoter [9]. (B) Protein A¢ reporter gene construct pEG998, earlier denoted pEG1000 [10], was used as cloning vector to subclone different AUG downstream context sequences using the restriction sites SwaI ⁄ SalI and SwaI ⁄ Csp45I.

E. I. Gonzalez de Valdivia and L. A. Isaksson Abortive translation due to drop-off at NGG codons

codons [10,21]. They also demonstrate the correlation between a negative effect on gene expression and a strong negative effect on growth of a pth– mutant strain.

Overexpression of tRNA has no effect on gene expression or growth inhibition associated with early NGG codons

including NGG,

The MB01 mutant strain is supposed to be growth inhibited at 37.5 (cid:1)C because of accumulation of pep- tidyl-tRNA, thus possibly causing decreased turnover and starvation for the tRNA moiety [22]. One would therefore expect that overexpression of the correspond- ing tRNA would protect from growth inhibition at 37.5 (cid:1)C by the +5 NGG codons, in a similar manner as does introduction of a plasmid encoding the pth+ gene. To test for this possibility a plasmid with +5 AGG in the test gene was combined in the MB01 strain with the plasmid pUBS520 that carries the tRNAArg4 gene, or plasmid pArgUW with the tRNAArg4 (decodes AGA and AGG) and tRNAArg5 (decodes only AGG) genes. A plasmid with a +5 GGG construct was combined with the plasmid pMO22 that carries the gene for the cognate tRNAGly1 (decodes only GGG).

pth+ carrying plasmid pVH1,

disturbed growth was the result of peptidyl-tRNA accumulation a plasmid (pVH1) containing the pth+ gene was introduced into the pth(Ts) strain, harbour- ing another compatible plasmid that carries the lacZ reporter gene with AGG or GGG at position +5. Transformants were streaked on broth plates, with or without 1 mm IPTG, and incubated at 30 (cid:1)C or 37.5 (cid:1)C. In the absence of a pth+ carrying plasmid, without IPTG induction, growth of the lacZ variant with AGG or GGG at position +5 was disturbed at 37.5 (cid:1)C (Table 3). In the presence of IPTG growth inhibition was more severe. Co-expression of the plas- mid pVH1 with its pth+ and the plasmid with the test gene with +5 AGG or GGG protected from the inhibitory effect by these codons at 37.5 (cid:1)C, even in the presence of IPTG. LacZ variants with CGG or UGG at +5 gave similar growth inhibition as AGG and GGG (not shown). These results strongly suggest that growth inhibition by +5 NGG codons at 37.5 (cid:1)C is the result of accumulation of peptidyl-tRNA, caused by drop-off from the ribosome during translation, and low activity of Pth in the MB01 mutant strain. Fur- thermore, no codons, inhibited growth of the MB01 strain if placed at +7 (Fig. 1D) even in the presence of IPTG. Also these results are consistent with an earlier report that there is little influence of +7 codons on gene expression [10,21]. The data presented here suggest that NGG codons at early coding positions cause growth inhibition of a pth(Ts) mutant strain as a result of peptidyl-tRNA drop-off and insufficient Pth activity.

Plasmid carrying cells with the different variants of +5 NGG codons were analysed in a similar test as described above for the pth+ plasmid. However, unlike the plasmid the pUBS520 (encoding tRNAArg4), pArgUW (encoding tRNAArg4 and tRNAArg5) or plasmid pMO22 (enco- ding tRNAGly1) did not protect cells from the negative effect by the induced test gene, with its +5 NGG codon AGG or GGG, respectively (Table 3). It was

Gene expression values in a normal pth+ strain, in the absence of IPTG induction, are presented as inserts in Fig. 1. These values confirm the previous findings of low gene expression being associated with early NGG

Table 3. Complementation of growth inhibition of MB01 by overexpressed genes. Plasmids and strains MG16555 or MB01 are described in Table 1. The strains were streaked on Luria–Bertani agar plates with IPTG (1 mM) (+) or without (–), and incubated at 37.5 (cid:1)C.

IPTG

– +

MG1655 MB01 MG1655 MB01 Plasmids Features

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++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ +++ +++ +++ +++ +++ ++++ +++ +++ +++ ++++ +++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ – pUBS250 pArgUW pMO22 pEG216 pEG216 ⁄ pVH1 pEG216 ⁄ pUBS250 pEG216 ⁄ pArgUW pEG207 pEG207 ⁄ pVH1 pEG207 ⁄ pMO22 – tRNAArg4 tRNAArg4; 5 tRNAGly 1 AGG +5 AGG +5 ⁄ pth+ AGG +5 ⁄ tRNAArg4 AGG +5 ⁄ tRNAArg4; 5 GGG +5 GGG +5 ⁄ pth+ GGG +5 ⁄ tRNAGly1 +++ – ⁄ + – ⁄ + +++ – ⁄ + ++++ – ⁄ + – ⁄ + – ⁄ + ++++ – ⁄ +

E. I. Gonzalez de Valdivia and L. A. Isaksson Abortive translation due to drop-off at NGG codons

the plasmids

encoding tRNAArg4 or noted that tRNAArg4 and tRNAArg5 were toxic for the MB01 mutant strain, but not for the wild-type strain, even in the absence of the lacZ variant with its +5 AGG codon. On the contrary, the plasmid that carries the tRNAGly1 gene did not alone influence growth of the MB01 mutant strain, nor did it suppress the low gene expression associated with +5 GGG.

two,

respectively,

tRNA supply was seen (Fig. 3B). The results suggest that overexpression of the tRNAs for the arginine codon AGG and the glycine codon GGG do not coun- teract the low gene expression caused by these codons low expression by an early at early positions. Thus, AGG and GGG is not suppressible by increased cog- nate tRNA to these respective codons or to the codon that precedes them. Both the arginine codons AGG and AGA are decoded by the same tRNAArg4. As shown in Fig. 3, in the presence or absence of tRNAArg4, AGA at positions +2, +3, +4, +5 or +7 is associated with high gene expression, thus being very from the related codon AGG. The other different NGG codons CGG and UGG were not analysed for the influence of increased concentration of cognate tRNAs.

The effect on gene expression by an increased intra- cellular pool of relevant tRNAs was analysed. For this purpose the A¢ reporter system was used (formally referred to as Z-protein) Fig. 2B. This reporter system is carried by a plasmid with the 3A¢ and 2A¢ genes [10]. Both are based on a gene which codes for three or identical engineered antibody binding B-domains of protein A from Staphylococcus aureus. Both genes are under separate control of iden- tical Ptrc promoters and Ttrp terminators. The 3A¢ test gene is used to analyse the effects on expression by base alterations whereas the 2A¢ is kept constant as an internal control. The protein products can be affin- ity purified in a single step using an IgG–Sepharose column. Gene expression can be estimated by separ- ation of the two A¢ proteins on gel electrophoresis. Scanning of the protein bands give the relative gene expression (3A¢ ⁄ 2A¢ ratio).

cognate decoding tRNAs

the

is

The question remains whether the plasmid associ- ated tRNA genes are expressed in the analysed bac- teria. For this reason, the levels of gene expression and tRNA concentration in a wild-type strain was com- pared using a plasmid encoding tRNAArg4 (decoding AGA ⁄ AGG) and tRNAArg5 (decoding AGG) and another plasmid with the 3A¢ test gene, using double antibiotic selection. As shown in Fig. 4, the protein expression value for +5 AGG is (cid:1) 10 times lower than that for +5 AGA, or compared to either codon at position +7. The low expression value for +5 AGG is not increased despite the fact that the concentration of significantly increased in the strain.

The question was asked if excess tRNA cognate to the codon preceding an NGG codon has any influence on the low gene expression associated with such codons. The lysine codon AAA, that gives high gene expression if located at position +2, was cho- sen. Plasmid constructs with AAA at +2 and either one of the NGG codons at +3 (Fig. 2B) were coex- pressed in strain XAC together with another plasmid encoding tRNALys. The transformants (pJMM19) with their two compatible plasmids were grown under appropriate double antibiotic selection pressure and the 3A¢ and 2A¢ protein products were isolated and quantified by SDS ⁄ PAGE. Figure 3A shows that tRNALys overexpression, being cognate to the +2 AAA, did not significantly suppress the low expres- sion caused by the +3 NGG codons in the 3A¢ test gene.

In summary, the NGG codons CGG, AGG, UGG and GGG are associated with very low gene expression if placed at positions +2 to +5. This phenomenon is not observed for GGN and GNG (where N is non-G) or for the other arginine codons CGU, CGC, CGA and AGA [10]. For AGG and GGG low expression is found even if the corresponding decoding tRNA is overexpressed in the cell. Peptidyl-tRNA drop-off is suggested to be the reason for the low expression val- ues observed for the analysed codons AGG and GGG at the early positions +2, +3 or +5. These codons appear not to give any significant drop-off if located at including the arginine +7. Other analysed codons, codons CGU and AGA, do not appear to give any drop-off at any location, as reflected by high expres- sion values and inability to inhibit growth of a pth(Ts) mutant strain.

Discussion

The single codons AGG, CGG, UGG and GGG (NGG) at positions +2 to +5 downstream of the initiation codon strongly decrease gene expression at

The question was also asked if excess tRNA, cog- nate to an early NGG codon, influences gene expres- sion. For this purpose, 3A¢ encoding plasmids with +2, +3, +4, +5 or +7 AGG, GGG or AGA were combined with plasmid pUBS250 (encoding tRNAArg4) or pArgUW (encoding tRNAArg4 and tRNAArg5) or plasmid pMO22 (encoding tRNAGly1). AAA was used as +2 codon in the 3A¢ constructs, where applicable, and it was also analysed at the other positions for comparison. No effect on gene expression by the extra

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E. I. Gonzalez de Valdivia and L. A. Isaksson Abortive translation due to drop-off at NGG codons

B

A

C

D

Fig. 2. Inhibition of growth of Pth(Ts) mutant strain MB01 by peptidyl-tRNA drop- off at 37.5 (cid:1)C. Growth of MB01 with differ- ent plasmid constructions. Open symbols; noninduced cultures; closed green symbols, cultures induced with IPTG (1 mM). Growing cultures were monitored by measurements of D590 as a function of time after the induc- tion with IPTG at time zero. The codons analysed (CGU, AGA, CGG, AGG, UGG or GGG) were placed at positions as indicated. Effects on lacZ gene expression, in the absence of IPTG induction, by the analysed codons in strain MC1061 are indicated in the insert. Noninduced b-galactosidase values are presented as Miller units [45]. (A) Codons at position +2. (B) Codons at position +3. (D) Codons at position +5. (C) Codons at position +7.

the initiation codon. If

the level of translation [9,10,23]. The effect is not seen for the other G-rich codons GGN or GNG (where N is non-G) or if the NGG codons are placed at the later positions +7 or +11. The negative effect by NGG requires that these bases constitute a codon and not merely an out-of-frame early base sequence in the mRNA [10]. Low gene expression has also been repor- ted for clusters of two [6,24,25] to five rare codons [26–29] in the early coding region.

Increased mRNA degradation is not

[9,10,21,27,29,30]. Chemical

likely to explain the low expression associated with early NGG codons footprinting of mRNA–ribosome complexes shows that at least 13

and possibly as many as 20 codons of mRNA are cov- ered by a single ribosome [31,32]. A second ribosome cannot start translation initiation as long as the first ribosome is still translating any codon in the early region downstream of the NGG codons in the downstream region are translated slowly down to, and including position +5, this would give extended pausing and thus low gene expression. However, pausing at position +7 should also interfere with ribosome loading at the translational start site even if the effect could possibly be smaller. Alternat- ively, if the codon is rapidly translated at position +7, a high gene expression should be the result. In this

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A

Fig. 3. Influence of overexpression of tRNA on gene expression associated with NGG codons. Cultivation of cells and induction by IPTG are as described in Material and methods. Absence (–) or presence (+) of tRNA is indicated below the figure, from top to bot- tom, with tRNA genes as listed correspondingly in the insert from left to right. Protein A¢ values were normalized and are presented as relative expression where 1.0 stands for 34 ± 0.015 units (3A¢ ⁄ 2A¢x100) [46]. The standard error of all experiments is ± 0.15 or smaller. (A) pJMM19 (encoding tRNALys) influences on expres- sion of the 3A¢ model gene with +3 NGG constructs. (B) Influence on 3A¢ gene expression by pUBS250 (encoding tRNAArg4), pArgUW (encoding tRNAArg4 and tRNAArg5) and pMO22 (encoding tRNAGly1) as combined with +2, +3, +4, +5 or +7 AGG, GGG, AGA or AAA at each indicated position in the 3A¢ gene.

B

tRNAArg4 and tRNAArg5, giving decoding of both AGA and AGG, has been reported to substantially suppress the low expression of a natural gene with sev- eral consecutive AGA and AGG codons [24,28,34,35]. Such overexpression has also been reported to suppress the inhibitory effect on a pth(Ts) mutant strain that is attributed to peptidyl-tRNA drop-off at these codons [19,36].

The codon dependent growth inhibition of the pth mutant strain MB01 described here requires that the test gene is induced by IPTG. The resulting growth inhibition can be suppressed by an introduced pth+ gene on a plasmid. This finding further supports the model that NGG at early positions gives drop-off and accumulation of peptidyl-tRNA thereby inhibiting growth of MB01, with its Pth deficient enzyme (Fig. 1). As the negative effect by NGG codons is not found for position +7, this suggests that a heptamin- opeptidyl-tRNA is stable on the translating ribosome, thus being prevented from drop-off, even if it decodes an NGG codon (Fig. 1D).

case, the question must be raised as to why early NGG should be decoded faster at position +7 than at position +5. It appears unlikely that ribosomal paus- ing at early NGG codons is the reason for the observed low gene expression.

Contrary to the observed suppression of AGG dependent growth inhibition by a pth+ gene in strain MB01 we did not obtain such suppression by supply- ing the AGG cognate tRNAArg4 or tRNAArg5 or both. These results are consistent and are obtained using tRNAArg4 and two different plasmids with the tRNAArg5 encoding genes. We find that the overex- pressed tRNAArg is toxic to the pth mutant, but not to the wild-type strain. We have no satisfactory explan- ation for the inability of overexpressed tRNAArg4 and tRNAArg5 to compensate for the deleterious effect by accumulated peptidyl-tRNA that is implied to take place in the pth(Ts) mutant strain. Apparently, the rea- son for its growth inhibition is not limiting tRNAArg. the overexpressed However, one possibility is that tRNA is undermodified and therefore less efficient in translation [37,38].

The length of the peptidyl moiety of the peptidyl- tRNA influences the drop-off process in the case of [16,33]. Overexpression of very small mini-genes

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and their respective cognate tRNAs (pUBS250, pAr- gUW or pMO22) did not influence gene expression lev- els. Thus, at least for the NGG codons AGG and GGG we fail to demonstrate any compensation of low gene expression by overexpressed tRNA. This is true either if the tRNA is cognate to the codon before or to the particular NGG codon itself, as analysed here.

The low expression values caused by early NGG co- dons are not increased in a strain with an inactive tmRNA system. Therefore, tmRNA activity is not the reason for the observed low gene expression by early NGG codon (not shown).

(A)

The translational ribosome complex is less stable in the beginning of a translated mRNA than further down [6,27,29]. The results presented here suggest an inefficient translational mechanism prone to a codon dependent peptidyl-tRNA drop-off at the very begin- ning of the coding region in mRNA. In the case of NGG codons located close to the initiation codon such instability appears to be even more pronounced. It could be speculated that during early translation the length of the nascent peptide is too short to reach the protein exit tunnel through the 50S subunit [16,33,42]. However, even though we find lowered expression in the cases of the arginine codons CGG and AGG, such effect is not observed for the other analysed arginine codons AGA, CGC, CGA and CGU. The amino acid sequence of the nascent peptide is the same in all of these cases. This fact eliminates any amino acid contri- bution by the amino acid residues in the nascent pep- tide to the observed low expression values observed for the NGG arginine codons. Similar arguments apply to the glycin codon family with GGG giving low while the others give high expression [10]. We are left with a model implying that the codon ⁄ anticodon interaction involving NGG codons is intrinsically weak in the early coding region, thus frequently leading to an abortive event like peptidyl-tRNA drop-off.

Fig. 4. tRNA levels and influence of pUBS250 (encoding tRNAArg4) on expression of the 3 A¢ reporter gene. The 3 A¢ gene carries [32P]ATP[cP]- AGG ⁄ AGA at position +5 or +7 as indicated. the labelled deoxyoligonucleotide probe which is specific for tRNAArg4 isoacceptor was used to identify and quantify the tRNA using slot blotting (columns 1–4 and 9–12). The tRNA values are presented as relative expression where 1.0 stands for wild-type value XAC (data not shown). Protein A¢ values (columns 5–8 and 13–16) were normalized and are presented as relative expression values, as explained in Fig. 3.

Experimental procedures

Chemicals and kits

Chemicals used were of the highest available purity from Sigma-Aldrich Chemie Gmbh (Steinheim, Germany). Restriction enzymes, T4 DNA ligase and T4 kinase were either from New England BioLabs (Ipswich, USA) Invitro- gen (Invitrogen AB, Sweden). Plasmids were prepared with GFXTM Micro Plasmid Prep Kit and gel band extractions were prepared with GFXTM PCR and Gel Band Purifica- tion Kit (Amersham Bioscience, Bucks, UK). DNA tem- plates were sequenced by MWG (The Genomic Company, Germany). Total RNA extraction was performed with

Overexpression of certain cognate tRNAs can give an increased gene expression [20,34,35,39–41]. Here, we have used the 3A¢ reporter gene system to analyse whe- ther the low gene expression associated with some early codons could be rescued by overexpression of the cog- nate tRNA. Co-expression of 3A¢ gene constructs with the lysine codon AAA at +2 together with a tRNALys encoding plasmid (pJMM19) in the strain, did not sup- press the low gene expression caused by any NGG codon at the following +3 location. Similarly, the coexpression of 3A¢ encoding plasmids with +2, +3, +4, +5 or +7 AGG, AGA or GGG in the 3A¢ gene

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(30%)

(Acrylamide ⁄ Bis

RNeasy Mini Kit (Qiagen, Valencia, CA; Branch Sweden). Oligonucleotides were labeled using [32P]ATP[cP] from Amersham Biosciences (Uppsala, Sweden). IgG Sepharose Fast Flow was from Amersham Biosciences. Electrophor- solution, esis Purity Reagent 29 : 1), Ammonium persulfate and TEMED were from Bio- Rad Laboratories AB, Sweden.

E. I. Gonzalez de Valdivia and L. A. Isaksson Abortive translation due to drop-off at NGG codons

Bacterial strains and plasmids

Escherichia coli strains used in this study are listed Table 1. The pth mutant strain MB01 was used to assay for pept- idyl-tRNA drop-off in vivo. Strain XAC [43] and plasmids pUBS250 (with tRNAArg4 gene), pArgUW (with tRNAArg4 and tRNAArg5 genes) pMO22 (with tRNAGly1 gene) or pJMM19 (with tRNALys gene) (Table 1) together with the protein 3 A¢ gene system (Fig. 1) was used to analyse for tRNA influences on gene expression. All tRNA encoding plasmids have different origins of replication and are com- patible with the lacZ or the 3 A¢ carrying plasmid.

Plasmid constructions and DNA sequencing

Plasmids were constructed using standard recombinant DNA techniques [44]. Some of the constructs (Table 1) have been reported previously [10].

the final concentrations: 100 lgÆmL)1 ampicillin, 50 lgÆmL)1 kanamycin and 10 lgÆmL)1 tetracycline. A 100-fold culture dilution was used as inoculum for growth in the same medium, and growth was followed by spectrophotometer measurements. For induction, IPTG (1 mm) was added at the mid-log phase of growth (D590 ¼ 0.2–0.25). Exponenti- ally growing cells (D590 ¼ 0.5) were cooled and harvested by centrifugation, followed by re-suspension in 1 mL 10 · TST buffer [46]. Cells were lysed by incubation at 95 (cid:1)C for 10 min, and cell debris was eliminated by centri- fugation. Protein A¢ was purified from the supernatant frac- tion using IgG–Sepharose (Pharmacia, Fairfield, CT, USA) mini-columns and a vacuum mini-fold system (Promega, Madison, WI, USA). A¢ proteins were eluted with 0.1 mL 0.5 m HAc at pH 3.2. The eluted protein was dried in a Savant SpeedVac(cid:2) plus SC110A (Telechem International, Inc. Sunnyvale, USA). Protein samples were dissolved in sample loading buffer, after denaturation at 95 (cid:1)C for 5 min. Separation of the A¢ proteins was achieved by SDS ⁄ PAGE (12% acrylamide) [44]. The bands were quanti- fied by scanning using FujiFilm Image Reader 1000 V1.2 (FujiFilm Life Science, Japan). The protein ratios were obtained by using image gauge 4.0 quantification pro- file ⁄ MW, microsoft excel program and extrapolation of plots. Protein A¢ values were normalized and are presented as relative expression where 1.0 stands for 34 ± 0.015 units (3 A¢ ⁄ 2 A¢x100) [46]. Each value represents the mean value of at least three independent measurements.

b-Galactosidase assays and growth conditions

Analysis of tRNA pools of unfractionated RNA immobilized by Northern hybridization slot blotting

Transformants were grown overnight at 37 (cid:1)C in minimal medium [45] supplemented with all amino acids at recom- [8] and 100 lgÆmL)1 ampicillin. mended concentrations These cultures were used to inoculate fresh medium at 37 (cid:1)C. Exponentially growing cells (optical density at 590 nm 0.4–0.5) were harvested without IPTG induction. b-Galactosidase activity of the lysed uninduced cells were determined [9]. All measurements were carried out using a Titer tech iEMS Reader MF (multiscan microplate photom- eter) and the Genesis computer program (Labsystems).

Peptidyl-tRNA drop-off experiments

MB01 cells with a pCMS71 derived plasmid (Fig. 1) were grown overnight at 30 (cid:1)C in M9 medium supplemented with all the amino acids at recommended concentration with and 100 lgÆmL)1 ampicillin [8,45]. Fresh cultures were grown until D590 ¼ 0.4 and split into two subcultures with an D590 ¼ 0.1 in fresh medium. One of the two cultures was inoculated together with IPTG (1 mm) and growth at 37.5 (cid:1)C was continued for a further 3.5 h.

Protein A¢ assay and growth conditions

(12–14 h, at 42 (cid:1)C)

Total RNA was extracted using RNeasy Mini Kit protocol. A 5-mL culture in minimal medium, supplemented with all amino acids [8] and the appropriate antibiotic, was induced with 1 mm IPTG at D590 ¼ 0.2. The bacteria (XAC with the appropriate plasmid) were then harvested at an D590 ¼ 0.5. The extracted total RNA was purified and denatured in 100 lL with a denaturing solution [50% (v ⁄ v) forma- mide, 7% (v ⁄ v) formaldehyde, 1· NaCl ⁄ Cit) and incubated at 68 (cid:1)C for 15 min. RNA (5 lg, [RNA] ¼ A260nm · dilu- tion · 40 lgÆmL)1) was blotted onto Hybond-XL nylon (Amersham Biosciences) using a manifold membrane apparatus from Hoefer Scientific. Total RNA was linked to the Hybond-XL nylon using ultraviolet light for 5 min. Oligonucleotides were labelled using [32P]ATP[cP] (Amer- shan Biosciences) and a T4 kinase kit (Invitrogen). To probe the tRNAArg4 the oligo 5¢-GAACCTGCGGCC- CACGACTTAGAA-3¢ was used for hybridization [34]. Probes were purified using MicroSpinTM G-25 columns (Amersham Pharmacia Biotech). The transferred RNAs to the were hybridized overnight [32P]ATP[cP] deoxyoligonucleotide probe, which is comple- mentary to the encoding tRNA sequence [44]. Filters were

Strains with plasmids were cultured overnight at 37 (cid:1)C in M9 medium [45] with all amino acids at recommended concentrations [8]. Antibiotics were added as necessary at

FEBS Journal 272 (2005) 5306–5316 ª 2005 FEBS

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1480 in the 16S ribosomal RNA affects expression of gln S. Nucleic Acids Res 19, 5247–5251.

9 Stenstro¨ m CM, Jin H, Major LL, Tate WP & Isaksson LA (2001a) Codon bias at the 3¢-side of the initiation codon is correlated with translation initiation efficiency in Escherichia coli. Gene 263, 273–284.

exposed to a phosphorimaging screen, scanned by image reader V1.8E software (FujiFilm FLA 3000) and saved using the image gauge V3.45 software (FujiFilm FLA 3000). The tRNA ratios were obtained by using Image Gauge 4.0 quantification profile ⁄ MW, microsoft excel program and extrapolation of plots, where 1.0 stands for tRNAArg4 for the wild-type strain value. Each value repre- sents the mean of at least three independent measurements.

E. I. Gonzalez de Valdivia and L. A. Isaksson Abortive translation due to drop-off at NGG codons

Acknowledgements

10 Gonzalez de Valdivia EI & Isaksson LA (2004) A codon window in mRNA downstream of the initiation codon where NGG codons give strongly reduced gene expres- sion in Escherichia coli. Nucleic Acids Res 32, 5198– 5205.

11 Kurland CG, Hughes D & Ehrenberg M (1996) Limita- tions of translational accuracy. In Escherichia coli and Salmonella thyphimurium: Cellular and Molecular Bio- logy (Neidhardt FC, ed.), pp. 979–999. ASM Press, Washington D.C.

12 Atherly AG & Menninger JR (1972) Mutant E. coli

We thank Dr Margarete Bucheli-Witschel for her input at the very beginning of this project. We thank Dr Isabella Moll, Dr Takayoshi Wakagi, Dr Valerie Heurgue-Hamard and Dr Michael O’Connor for gifts of plasmids and helpful advice. This work was suppor- ted by a grant from the Swedish Research Council to L.A. Isaksson.

strain with temperature sensitive peptidyl-transfer RNA hydrolase. Nat New Biol 240, 245–246.

References

13 Menninger JR (1976) Peptidyl transfer RNA dissociates during protein synthesis from ribosomes of Escherichia coli. J Biol Chem 251, 3392–3398.

14 Menninger JR (1979) Accumulation of peptidyl

tRNA is lethal to Escherichia coli. J Bacteriol 137, 694–696.

1 Roberts MW & Rabinowitz JC (1989) The effect of Escherichia coli ribosomal protein S1 on the transla- tional specificity of bacterial ribosomes. J Biol Chem 264, 2228–2235.

2 Spanjaard RA & van Duin J (1989) Translational reini-

tiation in the presence and absence of a Shine and Dalgarno sequence. Nucleic Acids Res 17, 5501–5507. 3 Stenstro¨ m CM, Holmgren E & Isaksson LA (2001b)

15 Hernandez-Sanchez J, Valadez JG, Herrera JV, Ontiveros C & Guarneros G (1998) lambda bar minigene-mediated inhibition of protein synthesis involves accumulation of peptidyl-tRNA and starvation for tRNA. EMBO J 17, 3758–3765.

16 Karimi R, Pavlov MY, Heurgue-Hamard V,

Cooperative effects by the initiation codon and its flank- ing regions on translation initiation. Gene 273, 259–265.

4 O’Connor M, Asai T, Squires CL & Dahlberg AE

(1999) Enhancement of translation by the downstream box does not involve base pairing of mRNA with the penultimate stem sequence of 16S rRNA. Proc Natl Acad Sci USA 96, 8973–8978.

Buckingham RH & Ehrenberg M (1998) Initiation factors IF1 and IF2 synergistically remove peptidyl-tRNAs with short polypeptides from the P-site of translating Escherichia coli ribosomes. J Mol Biol 281, 241–252. 17 Heurgue-Hamard V, Dincbas V, Buckingham RH & Ehrenberg M (2000) Origins of minigene-dependent growth inhibition in bacterial cells. EMBO J 19, 2701– 2709.

5 Moll I, Huber M, Grill S, Sairafi P, Mueller F, Brima- combe R, Londei P & Blasi U (2001) Evidence against an Interaction between the mRNA downstream box and 16S rRNA in translation initiation. J Bacteriol 183, 3499–3505.

6 Chen G-FT & Inouye M (1990) Suppression of the

18 Cruz-Vera LR, Hernandez-Ramon E, Perez-Zamorano B & Guarneros G (2003) The rate of peptidyl-tRNA dissociation from the ribosome during minigene expres- sion depends on the nature of the last decoding inter- action. J Biol Chem 278, 26065–26070.

19 Olivares-Trejo JJ, Bueno-Martinez JG, Guarneros G &

negative effect of minor arginine codons on gene expres- sion; preferential usage of minor codons within the first 25 codons of the Eschericia coli genes. Nucleic Acids Res 18, 1465–1473.

7 Looman AC, Bodlaender J, Comstock LJ, Eaton D,

Hernandez-Sanchez J (2003) The pair of arginine codons AGA AGG close to the initiation codon of the lambda int gene inhibits cell growth and protein synth- esis by accumulating peptidyl-tRNAArg4. Mol Micro- biol 49, 1043–1049.

20 Menez J, Heurgue-Hamard V & Buckingham RH

Jhurani P, deBoer HA & van Knippenberg PH (1987) Influence of the codon following the AUG initiation codon on the expression of a modified lacZ gene in Escherichia coli. EMBO J 6, 2489–2492.

(2000) Sequestration of specific tRNA species cognate to the last sense codon of an overproduced gratuitous protein. Nucleic Acids Res 28, 4725–4732.

8 Faxe´ n M, Plumbridge J & Isaksson LA (1991) Codon choice and potential complementarity between mRNA downstream of the initiation codon and bases 1471–

FEBS Journal 272 (2005) 5306–5316 ª 2005 FEBS

5315

dependent on the availability of the dnaY gene product. Gene 85, 109–114.

35 Zahn K (1996) Overexpression of an mRNA dependent

21 Croitoru VV, Bucheli-Witschel M & Isaksson LA (2005) In vivo involvement of mutated initiation factor IF1 in gene expression control at the translational level. FEBS Lett 579, 995–1000.

22 Menninger JR (1978) The accumulation as peptidyl-

on rare codons inhibits protein synthesis and cell growth. J Bacteriol 178, 2926–2933.

transfer RNA of isoaccepting transfer RNA families in Escherichia coli with temperature-sensitive peptidyl- transfer RNA hydrolase. J Biol Chem 253, 6808–6813.

36 Cruz-Vera LR, Magos-Castro MA, Zamora-Romo E & Guarneros G (2004) Ribosome stalling and peptidyl- tRNA drop-off during translational delay at AGA codons. Nucleic Acids Res 32, 4462–4468.

23 Stenstro¨ m CM & Isaksson LA (2002) Influences on

translation initiation and early elongation by the mes- senger RNA region flanking the initiation codon at the 3¢ side. Gene 288, 1–8.

24 Spanjaard RA & Duin JV (1988) Translation of the

37 Bjo¨ rk GR (1996) Escherichia coli and Salmonella typhimurium. In Escherichia coli and Salmonella thyphimurium: Cellular and Molecular Biology (Neidhardt FC, ed.), pp. 861–880. ASM Press, Washington D.C.

38 Gustafsson C, Govindarajan S & Minshull J (2004)

sequence AGG-AGG yields 50% ribosomal frameshift. Proc Natl Acad Sci USA 85, 7967–7971.

Codon bias and heterologous protein expression. Trends Biotechnol 22, 346–353.

39 O’Connor M (1998) tRNA imbalance promotes -1

25 Zahn K & Landy A (1996) Modulation of lambda inte- grase synthesis by rare arginine tRNA. Mol Microbiol 21, 69–76.

frameshifting via near-cognate decoding. J Mol Biol 279, 727–736.

26 Varenne S, Baty D, Verheij H, Shire D & Lazdunski C (1989) The maximum rate of gene expression is depen- dent on the downstream context of unfavourable codons. Biochimie 71, 1221–1229.

27 Rosenberg AH, Goldman E, Dunn JJ, Studier FW &

40 Imamura H, Jeon B, Wakagi T & Matsuzawa H (1999) High level expression of Thermococcus litoralis 4-alpha- glucanotransferase in a soluble form in Escherichia coli with a novel expression system involving minor arginine tRNAs and GroELS. FEBS Lett 457, 393–396.

41 Sorensen HP, Sperling-Petersen HU & Mortensen KK

Zubay G (1993) Effects of consecutive AGG codons on translation in Escherichia coli, demonstrated with a versatile codon test system. J Bacteriol 175, 716–722.

28 Chen G-FT & Inouye M (1994) Role of the

AGA ⁄ AGG codons, the rarest codons in global gene expression in Escherichia coli. Genes Dev 8, 2641–2652.

29 Gao W, Tyagi S, Kramer FR & Goldman E (1997)

(2003) Production of recombinant thermostable proteins expressed in Escherichia coli: completion of protein synthesis is the bottleneck. J Chromatogr B Analyt Tech- nol Biomed Life Sci 786, 207–214.

42 Yonath A & Berkovitch-Yellin Z (1993) Hollow, voids, gaps and tunnel in the ribosome. Curr Opin Struct Biol 3, 175–181.

Messenger RNA release from ribosomes during 5¢-trans- lational blockage by consecutive low-usage arginine but not leucine codons in Escherichia coli. Mol Microbiol 25, 707–716.

43 Coulondre C & Miller JH (1977) Genetic studies of the lac repressor. III. Additional correlation of mutational sites with specific amino acid residues. J Mol Biol 117, 525–567.

30 Jin H, Bjo¨ rnsson A & Isaksson LA (2002) Cis control of gene expression in E.coli by ribosome queuing at an inefficient translational stop signal. EMBO J 21, 4357– 4367.

44 Sambrook J, Fritsch EF & Maniatis T (1989) Molecular Cloning a Laboratory Manual. Cold Spring Harbor Laboratory Press, New York.

31 Beyer D, Skripkin E, Wadzack J & Nierhaus KH (1994) How the ribosome moves along the mRNA during protein synthesis. J Biol Chem 269, 30713–30717. 32 Green R & Noller HF (1997) Ribosomes and transla-

tion. Annu Rev Biochem 66, 679–716.

33 Dinc¸ bas V, Heurgue´ -Hamard V, Buckingham RH,

Karimi R & Ehrenberg M (1999) Shutdown in protein synthesis due to the expression of mini-genes in bacteria. J Mol Biol 291, 745–759.

45 Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, New York. 46 Bjo¨ rnsson A, Mottagui-Tabar S & Isaksson LA (1998) The analysis of translational activity using a reporter gene constructed from repeats of an antibody-binding domain from protein A. In Methods in Molecular Bio- logy, pp. 75–91. Edited by R. Martinª Human Press Inc, Totawa, NJ.

34 Brinkmann U, Mattes RE & Buckel P (1989) High-level expression of recombinant genes in Escherichia coli is

FEBS Journal 272 (2005) 5306–5316 ª 2005 FEBS

5316

E. I. Gonzalez de Valdivia and L. A. Isaksson Abortive translation due to drop-off at NGG codons