Competition between trans-translation and termination or elongation of translation.

Abstract

The effects of tRNA, RF1 and RRF on trans-translation by tmRNA were examined using a stalled complex of ribosome prepared using a synthetic mRNA and pure Escherichia coli translation factors. No endoribonucleolytic cleavage of mRNA around the A site was found in the stalled ribosome and was required for the tmRNA action. When the A site was occupied by a stop codon, alanyl-tmRNA competed with RF1 with the efficiency of peptidyl-transfer to alanyl-tmRNA for trans-translation inversely correlated to the efficiency of translation termination. The competition was not affected by RF3. A sense codon also serves as a target for alanyl-tmRNA with competition of aminoacyl-tRNA. The extent of inhibition was decreased with the length of the 3'-extension of mRNA. RRF, only at a high concentration, slightly affected peptidyl-transfer for trans-translation, although it did not affect the canonical elongation. These results indicate that alanyl-tmRNA does not absolutely require the truncation of mRNA around the A site but prefers an mRNA of a short 3'-extension from the A site and that it can operate on either a sense or termination codon at the A site, at which alanyl-tmRNA competes with aminoacyl-tRNA, RF and RRF.

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Competition between trans-translation and
termination or elongation of translation
Krisana Asano1, Daisuke Kurita1, Kazuma Takada2, Takayuki Konno2,3, Akira Muto1,2
and Hyouta Himeno1,2,*
1Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Science, Hirosaki University,
Hirosaki 036-8561,2The United Graduate School of Agricultural Sciences, Iwate University, Morioka 020-8550 and
3Department of Microbiology, AKITA Prefectural Institute of Public Health, Akita 010-0874, Japan
Received July 21, 2005; Revised and Accepted September 12, 2005
ABSTRACT
TheeffectsoftRNA,RF1andRRFontrans-translation
by tmRNA were examined using a stalled complex
of ribosome prepared using a synthetic mRNA
and pure Escherichia coli translation factors. No
endoribonucleolytic cleavage of mRNA around the
A site was found in the stalled ribosome and was
required for the tmRNA action. When the A site was
occupied by a stop codon, alanyl-tmRNA competed
with RF1 with the efficiency of peptidyl-transfer to
alanyl-tmRNA for trans-translation inversely correl-
ated to the efficiency of translation termination.
The competition was not affected by RF3. A sense
codon also serves as a target for alanyl-tmRNA
with competition of aminoacyl-tRNA. The extent of
inhibition was decreased with the length of the 30-
extensionofmRNA.RRF,onlyatahighconcentration,
slightly affected peptidyl-transfer for trans-transla-
tion, although it did not affect the canonical elonga-
tion. These results indicate that alanyl-tmRNA does
notabsolutelyrequirethetruncationofmRNAaround
the A sitebut prefers anmRNA ofashort30-extension
from the A site and that it can operate on either a
sense or termination codon at the A site, at which
alanyl-tmRNA competes with aminoacyl-tRNA, RF
and RRF.
INTRODUCTION
A special RNA called tmRNA or SsrA RNA has been found
in a wide variety of eubacteria and in some chloroplasts and
mitochondria (1–3). tmRNA having both tRNA and mRNA
properties plays an important role in a quality control system
during protein synthesis. When a ribosome stalls on a prob-
lematic mRNA, presumably at the 30end of a truncated mRNA
lacking a stop codon, tmRNA charged with alanine enters the
Asiteoftheribosometoactfirstasanalanyl-tRNA(4,5).Then
it acts as an mRNA to direct the addition of a short peptide tail
(6). This co-translation, known as trans-translation, terminates
at a stop codon that follows the tmRNA reading frame,
releasing tagged polypeptide and rescuingthe stalled ribosome
(7–9). The C-terminal tag-peptide is recognized by several
ATP-dependent proteases, resulting in degradation of tagged
polypeptide (10).Inaddition,trans-translation hasbeen shown
to facilitate the degradation of truncated mRNA by releasing a
stalledribosomeandthereby allowing 30-to50-exonucleasesto
access the 30end of free mRNA (11).
Since tmRNA comprises two functional domains, a tRNA
domain partially mimicking tRNA (12) and an mRNA domain
including the coding region for tag-peptide surrounded by
four characteristic pseudoknot structures (13), an elaborate
interplay of the two domains should be required for trans-
translation. Although the mode of tmRNA action for trans-
translation has been extensively studied, it has not been
clarified how tmRNA finds the ribosome of stalled translation.
Initially, the target for the tmRNA system has been assumed to
be the ribosome with a stalled translation in which the P site
but not A site is occupied by mRNA owing to the lack of
its 30-portion (7). Tandem rare codons on an artificial mRNA
can also be a target for trans-translation (14). Full-length and
C-terminally truncated polypeptides with a tag-sequence at the
C-terminus have also been identified as endogenous products
of trans-translation in the cell (15,16), and trans-translation
efficiently occurs at an inefficient UGA termination codon
preceded by rare arginine codons (17). These in vivo data
suggest that trans-translation can occur on either a sense
or nonsense codon at the A site when translation is stalled.
However, a recent finding of bacterial toxin that cleaves an
mRNA specifically at the A site in the ribosome stalled upon
amino acid starvation provides a new concept that mRNA of
stalled translation is targeted initially by these A site-specific
endoribonucleases and subsequently by alanyl-tmRNA for
trans-translation (18,19).
*To whom correspondence should be addressed. Tel: +81 172 39 3592; Fax: +81 172 39 3593; Email: himeno@cc.hirosaki-u.ac.jp
? The Author 2005. Published by Oxford University Press. All rights reserved.
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5544–5552
doi:10.1093/nar/gki871
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In prokaryotes, either RF1 or RF2 recognizes a stop codon
and catalyzes the hydrolysis of peptidyl-tRNA to release
the polypeptide from the ribosome (20). After this event,
RF3 stimulates the dissociation of RF1 or RF2 from the
ribosome (21), and then RRF, with the help of EF-G, allows
the ribosome to enter a new round of translation (22,23).
In the present study, a stalled complex of the ribosome
with a polyphenylalanyl-tRNA at the P site was prepared
from ribosomes programmed with synthetic mRNAs and
other pure translation factors from Escherichia coli. Using
this stalled complex of ribosome, the competition between
alanyl-tmRNA and RF1 at a nonsense codon was examined.
Ivanova et al. (24) have recently shown the competition at
a nonsense codon, and such a competition was also observed
in our system. We further examined the competition between
alanyl-tmRNA and valyl-tRNA or RRF at a sense codon with
different lengths of the 30-extension of mRNAs. We found that
alanyl-tmRNAcanenteranAsitefortrans-translationateither
the sense or termination codon with competition among
aminoacyl-tRNA, RF1 and RRF, even without cleavage of
mRNA at the A site. We also found that alanyl-tmRNA prefers
a shorter 30-extension of mRNA from the A site.
MATERIALS AND METHODS
Preparation of RF1
The gene encoding E.coli RF1, prfA, ligated into a pKK223-3
plasmid was cloned into E.coli strain JM109. Eight grams
of cells that overproduced RF1 were suspended in 16 ml of
buffer A [50 mM Tris–HCl (pH 7.5) and 10 mg/ml of 4-(2-
aminoethyl)benzenesulfonyl fluoride] and lysed by sonication.
The debris was removed by centrifugation at 9000 r.p.m.
(Hitachi R10A2) for 10 min, 20 000 r.p.m. (Hitachi P65A)
for 30 min and 80 000 r.p.m. (Hitachi RP80AT) for 30 min.
The supernatant was fractionated by DEAE–Sepharose col-
umn chromatography with a linear gradient of KCl from 0 to
200 mM in buffer A. The fractions containing RF1 were
pooled and applied to an Ni-NTA column (Pharmacia). RF1
was eluted by a linear gradient of imidazole from 0 to 20 mM.
The protein concentration was measured by Bradford’s
method using BSA as a standard (25). The purity was analyzed
by electrophoresis on an SDS polyacrylamide gel (26). The
polypeptide-chainreleasingactivityofRF1wasdeterminedby
measuring free formyl-[3H]methionine released from formyl-
[3H]methionyl-tRNAf
as described by Grentzmann and Kelly (27).
met?UUCAUGUAA?ribosome complex
Preparation of RF3
The gene encoding E.coli RF3, prfC, ligated into a pKK223-3
plasmid was cloned into E.coli strain JM109. Eight grams
of cells that overproduced RF3 were suspended in 16 ml of
buffer B [50 mM Tris–HCl (pH 7.5), 5 mM GDP and 10 mg/ml
of 4-(2-aminoethyl)benzenesulfonyl fluoride] and were lysed
by sonication. The debris was removed by centrifugation three
times at 9000 r.p.m. (Hitachi R10A2) for 10min, 20 000 r.p.m.
(Hitachi P65A) for 30 min and 80000 r.p.m. (Hitachi
RP80AT) for 30 min. The supernatant was then applied to
DEAE–Sepharose, and the proteins were eluted with a linear
gradient of KCl from 25 to 250 mM in buffer B. The fractions
containingRF3werepooled,adjustedtobepH6.0with20mM
sodium phosphate, and applied to a CM–Sepharose column
(Tosoh). RF3 was eluted with a linear gradient of NaCl from
0 to 200 mM. The GTPase activity of RF3 was measured in
the presence of ribosome. The hydrolysis of [32P]GTP into
[32P]GDP was measured by thin-layer chromatography (28).
Preparation of RRF
The gene encoding E.coli RRF, frr, ligated into a pKK223-3
plasmid was cloned into E.coli strain JM109. Eight grams of
cellsthatoverproducedRRFweresuspendedin16mlofbuffer
A and lysed by sonication. The debris was removed by cen-
trifugation at 9000 r.p.m. (Hitachi R10A2) for 10 min,
20 000 r.p.m. (Hitachi P65A) for 30 min and 80 000 r.p.m.
(Hitachi RP80AT) for 30 min. The supernatant was then
applied to Q-Sepharose Fast Flow (Pharmacia). The flow-
through fraction containing RRF was adjusted to be 1.5 M
ammonium sulfate and applied to Phenyl Sepharose 6 Fast
Flow (Pharmacia). RRF was eluted with a linear gradient of
ammonium sulfate from 1.5 to 0.5 M. The activity of RRF was
confirmed by monitoring the conversion from polysomes
to monosomes in the presence of EF-G by sucrose density
gradient centrifugation (23).
Preparation of tmRNA and SmpB
E.coli tmRNA was induced from an overproducing strain by
the addition of 1.0 mM Isopropyl-b-D-thiogalactopyranoside
(IPTG) and was purified as described previously (29). The
nucleic acid fraction was extracted with phenol and then
subjected to ethanol precipitation. After performing phenol
extraction and ethanol precipitation, the resulting fraction
was subjected to differential isopropylalcohol precipitations
to roughly remove DNA, followed by incubation with RNase-
free DNase I (Pharmacia). tmRNA was purified by electro-
phoresis on a 5% polyacrylamide gel containing 7M urea.
C-terminally His6-tagged SmpB was induced from an over-
producing strain by the addition of 1.0 mM IPTG and was
purified as described previously (30).
Preparation of the stalled complex of ribosome
Ribosomes were prepared from E.coli W3110 (28,31). E.coli
EF-G and EF-Tu with the addition of the sequence of His-tag
at the C-terminal were cloned and overproduced in E.coli.
They were purified using an Ni-NTA column (32,33) and
their ribosome-dependent GTPase activities were confirmed
by thin-layer chromatography (28).
E.coli
tRNAPhe
(Sigma)
[14C]phenylalanine by E.coli phenylalanyl-tRNA synthetase.
Polyphenylalanines were synthesized using ribosome pro-
grammed with synthetic mRNAs.Translation reaction mixture
(50 ml) contained 5 pmol ribosome, 100 pmol mRNA,
100 pmol EF-Tu, 3 pmol EF-G, 10 pmol [14C]phenylalanyl-
tRNAPhe,2mM spermidine, 1mM ATP, 0.2 mM GTP, 80 mM
Tris–HCl (pH 7.5), 30 mM ammonium chloride, 5 mM mag-
nesium acetate and 2.5 mM DTT. After different periods of
incubation at 37?C, a 10 ml aliquot of the mixture was with-
drawn and put in5ml of hot 5%trichloroacetic acid, preheated
at 90?C for 5 min. Then14C-labeled phenylalanines incorpor-
ated into a polypeptide, which were in the acid-insoluble frac-
tion, were quantified by using a liquid scintillation counter
(LSC-3500; Aloka). The phenylalanine incorporation was
wasaminoacylatedwith
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saturated by incubation at 37?C for 10 min, and the solution
was then used as a stalled complex of ribosome for further
studies.
Trans-translation and translation using the stalled
complex of ribosome
tmRNA was aminoacylated with [3H]alanine by alanyl-tRNA
synthetase from E.coli (4,34). Alanine was incorporated from
alanyl-tmRNA into a polyphenylalanine peptide chain by
incubating the stalled complex of ribosome with a tmRNA
mixture containing 5 pmol [3H]alanyl-tmRNA and 30 pmol
SmpB.Afterdifferentperiods ofincubationat37?C,analiquot
of the mixture was withdrawn and put in 5 ml of hot 5%
trichloroacetic acid to stop the reaction, and the radioactivity
of the acid-insoluble fraction was measured by using a liquid
scintillation counter.
E.coli tRNAVal(Sigma) was aminoacylated with [3H]valine
by valyl-tRNA synthetase from E.coli (35). Valine incorpora-
tion into polypeptide was determined by mixing the stalled
complex of ribosome with a valyl-tRNAValmixture containing
1 pmol [3H]valyl-tRNAVal, 0.1 pmol EF-G and 20 pmol
EF-Tuz. After incubation at 37?C for 30 s, an aliquot of the
mixture was withdrawn and put in 5 ml of hot 5% trichloro-
aceticacidtostopthereactionandtheradioactivityoftheacid-
insoluble fraction was measured by using a liquid scintillation
counter.
RESULTS
In vitro translation and peptidyl-transfer to
alanyl-tmRNA for trans-translation
Using pure translation factors, [14C]phenylalanine was
incorporated into polyphenylalanine from (UUC)10 or its
derivatives having a varying sequence of 30-extension. After
incubation of the mixture at 37?C for 10 min, the phenylalan-
ine incorporation was saturated (data not shown). The levels
of phenylalanine incorporation at 10 min from six kinds of
mRNAs were similar as shown in Figure 1A, and the solution
was used as a stalled complex of ribosome for further studies.
The stalled complex of ribosome was then incubated with
3H-labeled alanyl-tmRNA mixture. Alanine was gradually
incorporated only in the presence of SmpB, a tmRNA binding
protein (30,36), and the alanine incorporation was almost sat-
urated after 10 min of incubation (Figure 1B). During this
period, the amount of phenylalanines incorporated did not
change (Figure 1B, dotted lines). At a saturated time point, the
ratio of alanines to phenylalanines incorporated was ?0.09.
The alanine incorporation at 30 s from six kinds of mRNAs
is shown in Figure 1C. When mRNA having the longest
30-extension, (UUC)10GUA(A)12, was used, alanine incorp-
oration was significantly less than the alanine incorporations
using the other kinds of mRNAs.
Competition between RF1 and alanyl-tmRNA
The efficiency of termination depends on the stop codon and
its 30-adjacent (+4) nucleotide. UAAU and UAAG serve as
strong stop signals, while UGAC and UAGC give poor
termination efficiency (37). In the present study, two
different contexts of stop codons UAAU and UAGC that
are strong and weak stop signals for RF1, respectively,
were used as mRNAs to examine the competition between
RF1 and alanyl-tmRNA. Various amounts of purified RF1
were added to the tmRNA mixture with a fixed amount of
3H-labeled alanyl-tmRNA. Each of the tmRNA mixtures con-
taining various amounts of RF1 was incubated with the stalled
complex of ribosome at 37?C for 1 min. During this process,
phenylalanine incorporation was not affected by RF1. By
using mRNA without a stop signal sequence, RF1 had no
influence on the alanine incorporation by alanyl-tmRNA
(Figure 2). When using (UUC)10UAAU and (UUC)10UAGC
as mRNAs containing a stop signal for RF1, however, alanine
incorporation was significantly affected by RF1, the effect
being much greater when (UUC)10UAAU was used. When
using the same amounts of tmRNA and RF1 in the reaction,
the percentages remaining of alanine incorporation from the
stalled complexofribosome
(UUC)10UAAU and (UUC)10UAGC were 66 and 78%,
respectively. Apparently, the efficiency of peptidyl-transfer
for trans-translation in the presence of RF1 depended on
the preferenceofthestopsignalforRF1. These resultsindicate
that alanyl-tmRNA competes with RF1 at a termination codon
with the efficiency of trans-translation inversely correlated to
the efficiency of translation termination.
programmed with
RF3 did not affect the inhibition of RF1 on peptidyl-
transfer for trans-translation at a termination codon
RF3 has been shown to stimulate the dissociation of RF1 from
the ribosome after peptidyl-tRNA hydrolysis (21). It accentu-
ates the strength of the stop signal specified by the 4 nt stop
signal. Therefore, RF3 was added to the reaction mixture.
However, no significant difference was found between alanine
incorporation or between phenylalanine incorporation in the
presence of RF3 and absence of RF3 (Figure 3). This result
indicates that RF3 neither enhances nor inhibits the step of
peptidyl-transfer for trans-translation at a termination codon.
Competition between RRF and alanyl-tmRNA
The effect of RRF on alanine incorporation by tmRNA was
examined by the addition of RRF to the [3H]alanyl-tmRNA
mixture. After incubation of this mixture for 30 s with the
stalled complex of ribosome programmed with six kinds of
mRNAs, alanine incorporation was measured. Alanine incorp-
oration was decreased when the reaction mixture contained
0.5 and 5.0 mM of RRF, which were 10 and 100 times greater
than the amountoftmRNA, respectively(Figure4).Inthe case
of a smaller amount of RRF, no reduction of alanine incorp-
oration was detected (data not shown). The patterns of inhibi-
tion by RRF for all kinds of mRNAs were similar. Since
phenylalanine incorporation was not decreased by the addition
of RRF, the observed inhibition by RRF was not due to the
decrease in the stalled complex of ribosome. These results
indicate that alanyl-tmRNA competes with RRF to enter the
A site of the stalled ribosome either in the presence or absence
of mRNA in the A site.
Effect of alanyl-tmRNA on valine incorporation
TostudythecompetitionbetweentRNAandtmRNAatasense
codon, the effect of alanyl-tmRNA on the incorporation of
valine using mRNAs containing a GUA codon were observed.
After a saturating level of polyphenylalanine was synthesized
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as explained above, the [3H]valyl-tRNAValmixture was
added. After incubation at 37?C for 30 s, valine incorporation
wasmeasured.Asexpected,[3H]valinewassignificantlyincor-
porated when using (UUC)10GUA, (UUC)10GUA(A)6 or
(UUC)10GUA(A)12as an mRNA, while valine incorporation
was not observed when using (UUC)10UAGC lacking a valine
codon (Figure 5A). Similar levels of valine incorporation
were observed from three mRNAs having a GUA codon with
0
1000
(UUC)10
(UUC)10UAAU
(UUC)10UAGC
(UUC)10GUA
(UUC)10GUA(A)6
(UUC)10GUA(A)12
Phenylalanine incorporation (fmol)
(A)
0
10
20
30
40
50
60
70
80
90
100
0246810 121416 1820
Time (min)
0
200
400
600
800
1000
1200
(B)
Phenylalanine incorporation (fmol)
Alanine incorporation (fmol)
0
10
20
30
40
(UUC)10
(UUC)10UAAU
(UUC)10UAGC
(UUC)10GUA
(UUC)10GUA(A)6
(UUC)10GUA(A)12
(C)
Alanine incorporation (fmol)
Figure 1. In vitro polyphenylalanine synthesis directed by (UUC)10-based mRNAs and alanine incorporation by tmRNA from the stalled complex of ribosome.
(A) Fifty microliters of translation reaction mixture contained 5 pmol ribosome, 100 pmol mRNA, 200 pmol EF-Tu, 3 pmol EF-G, 10 pmol [14C]phenylalanyl-
tRNAPhe, 2 mM spermidine, 1 mM ATP, 0.2 mM GTP, 80 mM Tris–HCl (pH 7.5), 30 mM ammonium chloride, 5 mM magnesium acetate and 2.5 mM DTT. After
incubationat37?Cfor10min,a10mlaliquotwaswithdrawnfromthereactionmixtureandputin5mlofhot5%trichloroaceticacid.(B)Sixtymicrolitersofstalled
complexof ribosomes programmed with(UUC)10GUApreparedin (A) wasincubatedat 37?Cwith 20ml oftmRNAmixturecontaining4pmol[3H]alanyl-tmRNA
with (solid circle) or without (open circle) 60 pmol SmpB. After incubation at the indicated time point, a 10 ml aliquot was withdrawn and put in 5 ml of hot 5%
trichloroacetic acid. The incorporated alanines (solid line) and phenylalanines (dotted line) were quantified. (C) Fifteen microliters of stalled complex of ribosome
programmed with each kind of mRNA was incubated with 5 ml of tmRNA mixture containing 1 pmol pre-charged [3H]alanyl-tmRNA and 15 pmol SmpB. After
incubation at 37?C for 30 s, a 15 ml aliquot was withdrawn and put in 5 ml of hot 5% trichloroacetic acid. The incorporated alanines were quantified.
Nucleic Acids Research, 2005, Vol. 33, No. 17 5547

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differentlengthsofthe30-extensionfromtheGUAcodon.This
result indicates that aminoacyl-tRNA, unlike alanyl-tmRNA
(Figure 1C), enters the A site of a translating ribosome regard-
less of the length of the 30-extension of mRNA from the A site.
Various concentrations of alanyl-tmRNA with 15 pmol
SmpB were added to the [3H]valyl-tRNAValmixtures. These
mixtures were then combined with the stalled complex of
ribosome. After incubation at 37?C for 30 s, valine incorpora-
tions were measured (Figure 5B). At the same concentrations
of valyl-tRNAValand alanyl-tmRNA (50 nM), valine incorp-
oration was reduced by 10–20%. When the concentration of
alanyl-tmRNA was increased to 400 nM, 8 times higher than
that of valyl-tRNAVal, valine incorporation was decreased
more. Alanyl-tmRNA had the smallest inhibitory effect when
mRNA having the longest (12 nt) extension from the GUA
codon was used. These results indicate that the length of
30-extension of mRNA from the A site for alanyl-tmRNA is
critical for entrance into the stalled complex of ribosome more
than that for aminoacyl-tRNA.
RRF did not affect valine incorporation
Various concentrations of RRF were added to the [3H]valyl-
tRNAValmixtures. These mixtures were then mixed with the
stalled complexof ribosome. After incubationat 37?Cfor 30 s,
incorporation of [3H]valine was measured. No significant
change in valine incorporation from any type of mRNA used
was observed by the addition of 50 mM RRF, a 1000-fold
higher concentration than that of valyl-tRNAVal(Figure 6).
This result indicates that RRF neither enhances nor inhibits
substantially the entrance of aminoacyl-tRNA into the A site
of the translating ribosome.
Cleavage of mRNAs was not found from
the stalled complex of ribosome
mRNA labeled with32P at the 50end was used for synthesizing
the stalled complex of ribosome. Then mRNA in the stalled
complex of ribosome was examined by PAGE. To increase
the population of mRNA involved in the stalled complex of
ribosome, the concentrations of mRNA and ribosome in the
mixture of the stalled complex formation were decreased
and increased, respectively (Figure 7). In this system, >10%
0
100
0 0.51 1.52 2.5
RF1 (µM)
Alanine incorporation (%)
20
40
60
80
Figure 2. Effect of RF1 on alanine incorporation by tmRNA. Stalled complex
of ribosomes (30 ml) programmed with (UUC)10(circle), (UUC)10UAGC
(rectangle) or (UUC)10UAAU (triangle) mRNA was incubated in 10 ml of
tmRNA mixture containing 2 pmol pre-charged [3H]alanyl-tmRNA, 30 pmol
SmpBandvariousconcentrationsofRF1.Afterincubationat37?Cfor1min,a
10 ml aliquot was withdrawn and put in 5 ml of hot 5% trichloroacetic acid.
Alanine incorporation in the reaction without RF1 is regarded as 100%.
The experiments showed a good reproducibility and the error was within 10%.
Concentration of RF1 (µM)
Alanine incorporation (%)
Alanine incorporation (%)
Concentration of RF1 (µM)
Alanine incorporation (%)
(A)
(B)
(C)
0
100
00.2 0.4 0.60.81
0
100
00.20.40.6 0.81
0
100
0 0.2 0.40.6 0.81
Concentration of RF1 (µM)
80
60
40
20
80
60
40
20
80
60
40
20
Figure 3. Effect of RF1 on alanineincorporationby tmRNA in the presenceof
RF3. Stalled complex of ribosomes (30 ml) programmed with a (A) (UUC)10-
based mRNA (used as control), (B) (UUC)10UAGC or (C) (UUC)10UAAU
mRNAs was incubated with 10 ml of tmRNA mixture containing 2 pmol
[3H]alanyl-tmRNA,30pmolSmpB,variousconcentrationsofRF1and0(solid
circle)or 20pmolRF3 (opencircle).Afterincubationat 37?Cfor1 mina 10ml
aliquot was withdrawn and put in 5 ml of hot 5% trichloroacetic acid. Alanine
incorporation in the reaction without release factors is regarded as 100%.
The experiments showed a good reproducibility and the error was within 10%.
5548Nucleic Acids Research, 2005, Vol. 33, No. 17