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 h
Trang 1Abortive 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
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
for gene expression, presumably at the translational level
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
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
Abbreviations
SD, Shine–Dalgarno; DR, downstream region; Pth, peptidyl-tRNA hydrolase; IPTG, isopropyl thio-b- D -galactoside; Ts, temperature sensitive.
Trang 2mRNA levels or secondary structure formation.
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
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
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]
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]
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
Results
Induction of a test gene with an early NGG
codon inhibits growth of a pth(Ts) strain
Codons at position +2, immediately following the
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]
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) This complementation is not complete, probably because of the low copy number of pVH1 [20]
Table 1 Bacterial strains and plasmids.
Relevant characteristics References Strains
MC1061 araD139, D(lacI POZYl) 74, galU,
galK, rpsL, D(ara A BC-leu)7697, hsdR, mcrB, Sm S ,
[44]
XAC D(lacproB), arg E, ara, gyrA, rpoB, thi [43]
MB01 MG1655, zdh-925::Tn10, pth(Ts) This work Plasmids
lacZ pCMS71 Derivative of pCM21 with cloning
cassette, Amp R
[9,10] Protein A¢
pHN109 Derivative of pAB93 with
an internal 2A¢
[30]
Control gene and cloning cassette, Amp R
pEG998 Derivative of pHN109 with Csp45I
restriction site
[10]
Others pUBS250 arg U + (tRNA Arg4 ), Kan R [34] pArgUW argU + (tRNA Arg4 ), argW + (tRNA Arg5 ) Kan R [40]
pJMM19 lysV + (tRNA Lys ), Kan R [20] pMO22 glyU + (tRNA Gly1 ), Tet R [39]
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 m M ) (+) or without (–), and incubated at the indicated temperatures.
Strains Plasmid
30 C 37.5 C 43 C 30 C 37.5 C 43 C
Trang 3To 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 30C,
followed by dilution into the same medium with or
without isopropyl thio-b-d-galactoside (IPTG; 1 mm)
whereafter growth was continued at 37.5C 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
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
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 43C and it showed disturbed growth at 37.5C (Table 2) To confirm that the
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.
Trang 4disturbed 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 30C or
37.5C 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
37.5C (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 37.5C, 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 37.5C
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, including NGG, 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
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
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
The MB01 mutant strain is supposed to be growth inhibited at 37.5C 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 37.5C 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)
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 pth+ carrying plasmid pVH1, the plasmid 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 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 m M ) (+) or without (–), and incubated at 37.5 C.
IPTG
pEG216 ⁄ pArgUW AGG +5 ⁄ tRNA Arg4; 5
pEG207 ⁄ pMO22 GGG +5 ⁄ tRNA Gly1
Trang 5noted that the plasmids encoding tRNAArg4 or
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
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 two, respectively, 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)
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
(pJMM19) encoding tRNALys The transformants
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
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
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
at early positions Thus, low expression by an early 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 different from the related codon AGG The other NGG codons CGG and UGG were not analysed for the influence of increased concentration of cognate tRNAs
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 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 the cognate decoding tRNAs is significantly increased in the strain
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 +7 Other analysed codons, including the arginine 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
Trang 6the 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 likely to
explain the low expression associated with early NGG
codons [9,10,21,27,29,30] Chemical 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 initiation codon If 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
Fig 2 Inhibition of growth of Pth(Ts)
mutant strain MB01 by peptidyl-tRNA
drop-off at 37.5 C Growth of MB01 with
differ-ent plasmid constructions Open symbols;
noninduced cultures; closed green symbols,
cultures induced with IPTG (1 m M ) Growing
cultures were monitored by measurements
of D 590 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.
Trang 7case, 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
The length of the peptidyl moiety of the
peptidyl-tRNA influences the drop-off process in the case of
very small mini-genes [16,33] Overexpression of
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)
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 tRNAArg4or tRNAArg5or both These results are consistent and are obtained using two different plasmids with the tRNAArg4 and tRNAArg5 encoding genes We find that the overex-pressed tRNAArgis toxic to the pth mutant, but not to the wild-type strain We have no satisfactory explan-ation for the inability of overexpressed tRNAArg4and 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 However, one possibility is that the overexpressed tRNA is undermodified and therefore less efficient in translation [37,38]
A
B
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¢ x 100) [46] The standard error of all experiments is ± 0.15
or smaller (A) pJMM19 (encoding tRNA Lys ) influences on expres-sion of the 3A¢ model gene with +3 NGG constructs (B) Influence
on 3A¢ gene expression by pUBS250 (encoding tRNA Arg4 ), pArgUW (encoding tRNA Arg4 and tRNA Arg5 ) and pMO22 (encoding tRNA Gly1 )
as combined with +2, +3, +4, +5 or +7 AGG, GGG, AGA or AAA at each indicated position in the 3A¢ gene.
Trang 8Overexpression 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 exsup-pression 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
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)
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
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 (InvitroInvitro-gen 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
Fig 4 tRNA levels and influence of pUBS250 (encoding tRNAArg4)
on expression of the 3 A¢ reporter gene The 3 A¢ gene carries
AGG⁄ AGA at position +5 or +7 as indicated (A) [ 32
P]ATP[cP]-labelled deoxyoligonucleotide probe which is specific for the
tRNA Arg4 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.
Trang 9RNeasy 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-esis Purity Reagent (30%) (Acrylamide⁄ Bis solution,
29 : 1), Ammonium persulfate and TEMED were from
Bio-Rad Laboratories AB, Sweden
Bacterial strains and plasmids
Escherichia colistrains 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 tRNAArg4gene), 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]
b-Galactosidase assays and growth conditions
Transformants were grown overnight at 37C in minimal
medium [45] supplemented with all amino acids at
recom-mended concentrations [8] and 100 lgÆmL)1 ampicillin
These cultures were used to inoculate fresh medium at
37C 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 30C in M9 medium supplemented
with all the amino acids at recommended concentration
with and 100 lgÆmL)1ampicillin [8,45] Fresh cultures were
grown until D590¼ 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
37.5C was continued for a further 3.5 h
Protein A¢ assay and growth conditions
Strains with plasmids were cultured overnight at 37C in
M9 medium [45] with all amino acids at recommended
concentrations [8] Antibiotics were added as necessary at
the final concentrations: 100 lgÆmL)1ampicillin, 50 lgÆmL)1 kanamycin and 10 lgÆmL)1tetracycline 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 (D590¼ 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
95C 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 plus SC110A (Telechem International, Inc Sunnyvale, USA) Protein samples were dissolved in sample loading buffer, after denaturation at 95C 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
Analysis of tRNA pools of unfractionated RNA immobilized by Northern hybridization slot blotting
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 D590¼ 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 68C for 15 min RNA (5 lg, [RNA] ¼ A260nm · dilu-tion · 40 lgÆmL)1) was blotted onto Hybond-XL nylon membrane (Amersham Biosciences) using a manifold 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 were hybridized overnight (12–14 h, at 42C) to the [32P]ATP[cP] deoxyoligonucleotide probe, which is comple-mentary to the encoding tRNA sequence [44] Filters were
Trang 10exposed 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
tRNAArg4for the wild-type strain value Each value
repre-sents the mean of at least three independent measurements
Acknowledgements
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
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