nuclease m in the processing of the messenger for the (3(' subunits of RNA polymerase is proposed. Key words: polynucleotide phosphorylase/ribonuclease In/.
The EMBO Journal vol.6 no.7 pp.2165-2170, 1987
The first step in the functional inactivation of the Escherichia coli polynucleotide phosphorylase messenger is a ribonuclease Ill processing at the 5' end
C.Portier, L.Dondon, M.Grunberg-Manago and P.Regnier Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France Communicated by M.Grunberg-Manago
The transcripts covering pnp, the gene encoding polynucleotide phosphorylase, are processed by ribonuclease Im. In this study, it is shown that the steady state level of the pnp mRNA increased 11-fold in a ribonuclease HI-deficient strain. The synthesis rate of this messenger is only slightly affected in the mutant strain whereas the half-life, which is 1.5 min in the wild type, is considerably increased to more than 40 min. Moreover, polynucleotide phosphorylase is 10-fold overexpressed in the mutant strain, which shows that unprocessed pnp mRNA is functional. The position of the ribonuclease im-sensitive site suggests that the sequence involved in the stabilization of the pnp mRNA is located at the 5' end of the message and that the RNase HI processing triggers the decay of the transcripts downstream. A similar function for ribonuclease m in the processing of the messenger for the (3(' subunits of RNA polymerase is proposed. Key words: polynucleotide phosphorylase/ribonuclease In/ messenger stabilization/mRNA decay/RNA polymerase Introduction Although it has been known for some time that the inherent instability of mRNA in prokaryotic organisms plays an important role in the rapid adaptation of microorganisms to changing growth conditions, little is known about how this regulation is brought about. Several different models describing mRNA degradation have been proposed (Gegenheimer and Apirion, 1981; Panayotatos and Truong, 1985; Belasco et al., 1986; Cannistraro et al., 1986; Kennell, 1986; Newbury et al., 1987). Some of these essential characteristics can be summarized as follows: first, the mRNA is efficiently protected against degradation by translating ribosomes. Second, in polycistronic mRNA, cleavages occur between coding regions and the decay does not necessarily follow an exclusively unidirectional wave (e.g. 5' to 3') (Kepes, 1967; Blundeil et al., 1972; Achord and Kennell, 1974; Schlessinger et al., 1977; Cannistraro et al., 1986). Third, for individual messages, each mRNA decays at a unique rate such that the halflife varies from 40 s to -20 min (Pedersen and Reeh, 1978). Fourth, the differences in mRNA stabilities have been accounted for by differences in translation efficiency and/or by the presence of structural determinants located near either the 5' or 3' extremities of the message. Fifth, the decay of the transcripts involves limited cleavages by endonucleases and participation of 3' exonucleases. Sixth, the direction of decay of a single message within the molecule is 3' to 5' (von Gabain et al., 1983; Newbury et al., 1987) or 5' to 3' (Cannistraro and Kennell, 1985). The specific components involved in mRNA degradation have not been identified. No ribonuclease mutants producing a slower IRL Press Limited, Oxford, England
functional decay of mRNA have been isolated. Indeed, there is no evidence that any specific nuclease with a defined target is involved. No 5' exonuclease has been described and other 3' to 5' processive exonucleases are presumably involved only in mass decay of messengers. Data obtained with bacteriophages have shown that RNase Ill plays a role in the processing of mRNA. RNase III cleavage of T7 early phage mRNA (Saito and Richardson, 1981) enables it to be translated. On the other hand, for X phage, RNase Ill cleavage was shown to provoke the decay of mRNA (Lozeron et al., 1977; Schmeissner et al., 1984). These two examples are quite opposite in effect since RNase HI cleavage produces stable T7 mRNA but allows the rapid decay of X mRNA. In E. coli, the role of RNase IH in the process of stable RNA is well known, but no clear effect on mRNA has been described. However, two polycistronic mRNAs are known to carry RNase HI-sensitive sites. They are the rpUL - rpoBC operon, encoding ribosomal protein L10, L12, and (3(3' subunits of RNA polymerase (Barry et al., 1980) and the rpsO-pnp operon, encoding the ribosomal protein S 15 and polynucleotide phosphorylase (Portier and Rdgnier, 1984; Takata et al., 1985; Rdgnier and Portier, 1986). The striking common feature of these two operons is that they have an RNase III site in the intercistronic space separating the ribosomal protein gene(s) from the downstream gene(s). The transcripts of these operons are very rapidly processed by RNase HI in wild type strains (nc+), whereas a cotranscript of the whole operon was observed in the RNase Ill deficient isogenic strain (mc-) (Barry et al., 1980; Regnier and Portier, 1986). To date, no function for the RNase III processing site in the (3(' subunits messenger has been found (Dennis, 1984). In an effort to get more information on the role of RNase HI in RNA stability, we have analysed the effect of RNase III on the expression of the rpsO-pnp operon. This has been done by measuring both the rates of pnp mRNA synthesis and degradation, and also the relative levels of pnp mRNA and polynucleotide phosphorylase.
Results The steady-state level of pnp mRNA in rnc+ and mc- strains The steady-state level of a mRNA reflects both its rate of synthesis and its rate of decay. Thus, if RNase III cleavage affects either one of these processes, a modification of the steady-state level of pnp mRNA should be observed. Previous studies have shown that pnp mRNAs are synthesized from two promoters (PI and P2) and are processed by several cleavages in the intercistronic part of the message (Rdgnier and Portier, 1986; R6gnier et al., 1987). The different pnp transcripts previously identified by SI mapping in mnc+ and mcstrains are shown in Figure 1. In order to estimate the relative amounts of the different pnp mRNAs, Northern blot hybridizations were carried out using the 1.7 kb SacH-MluI DNA fragment covering 81 % of the pnp 2165
C.Portier et al.
A
RIII p1
rpILfi~
rplJ
y
r-"
mm
I
AI I
t2
rpo C
rpo B
r~
¶
~~~~~~~~~~~~~~~~IsIII a
9 I'Ia
a
0.5 kb
B
jIILI
tiY2
p np
Ui
U
PstlI Bg Il
0.2 kb
post I
Sma I
m Mlu I
Sac 2
1
+10
2
__
4
Fig.
..... [..
1. Structural
organization of rpsO-pnp and rplJL-rpoBC operons. Coding sequences, transcription signals and maturation site in both operons are P1P2, promoters; t1t2, termtinators, RIII, RNase III-sensitive site; horizontal arrows give the direction of transcription; open boxes represent the structural part of the genes. Black boxes beneath the map correspond to the probes used in hybridization experiments (see text); cleavage sites at each extremity are specified. Transcripts previously identified by SI mapping in the rpsO-pnp operon are represented by wavy lines. (A) rplJL-rpoBC operon; (B) rpsO-pnp operon. shown.
as probe (see Figure 1). In the wild type strain, transcripts are 2200 nucleotides in length which fits with the size expected for processed molecules extending from the RNase IHI sensitive site to the terminator t2 (2250 nucleo-
mc& strains. This gene was chosen because it is well studied (Springer et al., 1985) and its mRNA contains no known potential RNase HII processing sites. Northern blot analysis showed
tides) (Figure 2, lanes 1, 2 and 3). Since it
(data
structural gene
most pnp
no
differences in size
or
quantity
of thrS mRNA in the two strains
previously shown that RNase IHI completely cleaves all the mRNAs spanning the processing site (R6gnier and Portier, 1986), but that there is appreciable readthrough at t2 (Rdgnier et al., 1987), it is probable that the minor 2700 nucleotide transcript, clearly resolved in the gel, extends to about 500 nucleotides beyond the terminator t2 (Figure 1, transcript 4). In the RNase HII-deficient strain, the main transcripts are slightly larger than in the wild type (Figure 2, lanes 4, 5 and 6). It is about 2400 nucleotides in length and corresponds well in size to the RNase HI unprocessed transcripts 2 and 3 shown in Figure 1. The 2900 nucleotide minor band very likely includes rpsO-pnp cotranscripts initiated at PI and terminated at t2 (2790 nucleotides) together with transcripts initiated at P2 which
synthesis are very similar in the mc+ and a slight increase in the apparent rate of pnp nmRNA synthesis in the mc6 strain. However, this increase is too small to account for the II1-fold increase in steady-state
terminate
pnp mRNA level.
nucleotides
500
was
downstream
of the
terminator
t2
(about 2800 nucleotides). In addition to
generating
Moreover, the
11I
most of
presence
monocistronic
of
times
more
pnp mRNA in the mutant strain.
mcG
strain is due to
2400
nucleotide pnp
the pnp mRNA in the
high amounts transcript.
of the
mutation on the verify that the effect of the mcG rpsO-pnp operon is not a general phenomenon, the levels of threonyl-tRNA synthetase nmRNA were measured in mnc' and To
2166
Relative
mc-
shown).
rates
of synthesis
and
decay of pnp
mRNA in
To determine whether the effect of the
mc
mRNA levels
or
were
the level of
was at
carried out to
synthesis rate, for 2 mmn with pnp
mc+
and
strains
measure
mRNA of [3
synthesis
mutation
on
pnp
decay, experiments
both parameters. To determine the
mc-
and
mc+
H]uridine,
specific probes.
was
strains, pulse-labeled
filter-hybridized
with thrS and
The data of Table I show that the rates of
pnp and thrS mRNA
mc&
strains. There is
The rate of
somewhat
longer different transcripts, the absence of RNase IHI has a striking effect on the amount of pnp mRNA (Figure 2). Densitometric analysis of the autoradiograph and of other similar experiments allow us to estimate that there is about
not
pulse-labeled thrS and pnp mRNA were by blocking mRNA synthesis with rifampicin and nalidixic acid and chasing with cold uridine. It was first shown that the mc mutation does not affect significantly total mRNA degradation by verifying that, under these conditions, the percentage of the radioactivity incorporated decreased decay
of
measured in the two strains
at
about the
mRNA
same rate
(data
not
in
shown).
mc+
very different in the two strains mRNA to vary
strain
was
also estimated in the
from 2
mmn
in the
(Figure 3). Thus,
and
mc6
strains for the bulk of
For thrS mRNA, the half-life
( -4
mmi).-
samne experiments
mc+
strain to 4
the presence of the
was not
The half-life of and
min
mc-
was
in the
SiS5
found
mc-
mutation pro-
RNase m in mRNA decay
100
50
a
20
iI 4
0) () (
ES 10 L
4
Z 5
1
3
5
7
9
11
3
1
Ti
Fig. 2. Intracellular concentration of pnp mRNA. Equal amounts of total RNA (12.5 /ig, lanes 1 and 4; 25 jig, lanes 2 and 5; 50 M4g, lanes 3 and 6) prepared from rnc+ (lanes 1, 2 and 3) and rc- (lanes 4, 5 and 6) strains were separated on a denaturing agarose gel, blotted and probed with 32plabelled SacII-MluI DNA fragment from the pnp gene. RNA size markers (Gibco BRL) were run on a lane of the same gel which was cut out before blotting and stained with ethidium bromide. Positions and lengths of these RNA are indicated on the left of the picture in small characters. Sizes of the main pnp transcripts, deduced from their relative migration rates, are indicated in large characters. Other faint bands also appear on the autoradiograph whose origin is not discussed in this article because they correspond to very minor amounts of pnp mRNA. Some are long mRNA which are either initiated at promoter(s) upstream of the P1 rpsO promoter (R6gnier and Portier, 1986) or are readthrough transcripts extending downstream of t2 (Rdgnier et al., 1987). Rapidly migrating minor mRNA, about 1 kb in length, which is too short to encode polynucleotide phosphorylase, are probably intermediary products of pnp mRNA degradation.
duced only a limited increase in rpsO mRNA. A similar small effect has been previously observed on specific mRNA half-lives in the rnc- strain (Talkad et al., 1978). However for pnp mRNA, a large difference was observed: a half-life of 1.5 min in the rnc+ strain is converted to quasi-stability for the mcstrain (half-life estimated at greater than 40 min) (Figure 3). These results show that the lack of RNase Ill cleavage has a specific and pronounced effect on the stability of pnp mRNA. This effect is quantitatively more important than the 1.6-fold increase in pnp mRNA synthesis rate observed and is sufficient to account for the 11-fold enhancement in pnp mRNA concentration in the mc- strain.
Polynucleotide phosphorylase levels in mc+ and mc- strains Since there is an accumulation of pnp mRNA in the mc- strain, we next asked the question whether this mRNA is functional, i.e. whether it is capable of being translated into polynucleotide phosphorylase. The specific activities of polynucleotide phosphorylase and of threonyl-tRNA synthetase were determined in crude extracts of exponential cultures of rnc+ and mc- strains. Very little dif-
5
me
7
9
11
1
3
5
7
9
11
( min )
Fig. 3. Decay of specific mRNA in rnc+ and rc- strains. Exponential cultures of BL322 and BL321 were pulse-labelled for 1 min with [3H]uridine and then RNA synthesis was stopped by rifampicin, nalidixic acid and cold uridine as described in Materials and methods. At given times, samples were withdrawn. Purified RNAs were extracted and hybridized with the gene-specific probes. For each probe, the percentage of mRNA remaining was calculated from the net radioactivity hybridized at a given time divided by the maximum radioactivity hybridized. The maximum counts/min (100% value) hybridizing to thrS, rpsO and pnp probes are respectively 505, 1508 and 2370 in BL322 strain and 1084, 669 and 2956 in BL321 strain. Open symbols, BL322; filled in symbols, BL321, ThrS; threonyl-tRNA synthetase mRNA, S15; ribosomal protein S15 mRNA. PNP; polynucleotide phosphorylase mRNA.
Table I. Synthesis rates of pnp and thrS mRNAa Strains BL32 (rnc+) BL321 (mc-)
%/Nucleotide x10-5 thrS (control)
pnp
1.48 1.67
1.70 2.8
'Each assay consists of three separate hybridizations where the input RNA was increased in the ratio 1-24. The net count per filter was obtained by subtracting the non-specific counts bound to the control filters. The %/nucleotide represents the average fraction of input radioactivity in specific RNA:DNA hybrids divided by the number of specific nucleotides in the DNA probe.
ference is observed in the specific activity of the threonyl-tRNA synthetase in the two strains (Table II). On the other hand, the specific activity of polynucleotide phosphorylase increased 10-fold in the rnc- strain. Thus, the increase in messenger level produces a proportional increase in its translation product, which demonstrates that accumulated pnp mRNA is as equally functional as the processed transcript. ,B and (3' subunits of RNA polymerase are also overexpressed Since overexpression of a protein is accounted for by the higher amount of its messenger, proteins overproduced in an mcstrain might be derived from an increase of unprocessed mRNA. This can be easily verified by examining the production of the p3(' subunits of RNA polymerase whose mRNA has been demonstrated to be processed by RNase III.
2167
C.Portier et at.
Table H. Specific activity of polynucleotide phosphorylase and threonyltRNA synthetase in rnc+ and rc- strains Strains
BL322 (rnc+) BL321 (rc-) Ratio of specific activity
Specific activity Polynucleotide phosphorylase 2.5
25.9 10.4
Threonyl-tRNA synthetase (control) 432
620 1.4
When total proteins of the two strains are separated on a 12.5% SDS polyacrylamide gel, clear differences in the pattern of bands were detectable. Several proteins are overproduced in the mcextract and some are missing (Figure 4). Among the overexpressed proteins, which include PNPase, are the fif' subunits of RNA polymerase which can be easily identified by comparison with the migration of purified enzymes (data not shown). However, this overproduction, although unambiguous, is quantitatively less than that of polynucleotide phosphorylase. Discussion The results presented here clearly establish that polynucleotide phosphorylase and its mRNA are both overproduced in a strain deficient for RNase III. This overproduction is shown to be the result of a specific stabilization of the pnp mRNA which retains a full translational capacity. We have previously shown that a RNase IEI-sensitive site exists at the 5' end of the pnp monocistronic transcripts, upstream of the initiation codon of the polynucleotide phosphorylase, and that in the wild type strain, all transcripts covering pnp are cleaved at this site. Pnp mRNAs in mc- strains are somewhat longer and it is these longer species which accumulate. Thus, in the mc strain, the transcripts are longer and stable whereas in the rnc+ strain, the transcripts are all processed and unstable. So the logical conclusion is that cleavage by RNase Ill triggers pnp mRNA decay. The simplest explanation of this phenomenon is that a structural determinant involved -in the stabilization of this mRNA is removed by the RNase Ill processing. Whether it is the region upstream of the RNase I1 site which is involved in this mechanism or the duplex stem of the RNase HI site itself remains to be determined. Stem-loop structures are believed to contribute to mRNA stability because they are presumed to be more resistant to ribonuclease attack (Schmeissner et al., 1984; Kennell et al., 1986; Newbury et al., 1987). However, factors other than secondary structure might contribute to mRNA stability (Gorski et al., 1985; Belasco et al., 1986). The structural determinants which have been claimed as stabilizing influences are located at either end of the messenger. This implies that degradation can start either at the 5' or 3' end depending on the RNA species. For example, Newbury et al. (1987), have shown that for malE and hisJ, the presence of the repetitive extragenic palindromic sequence 'REP' at the 3' end of these mRNAs stabilizes the upstream RNA and that, for these mRNAs, degradation normally occurs in a 3' to 5' direction. On the other hand, Cannistraro and Kennell (1985) propose that lacZ mRNA is degraded in a 5' to 3' direction. In the case described in this paper, cleavage by the RNase IH initiates pnp mRNA degradation at the 5' end which presumably is the first step of a 5' to 3' decay. The steps involved in this latter process are unknown. Perhaps, after cleavage at the RNase IH site, a refolding of the 5' extremity hinders ribosome binding 2168
Fig. 4. Polyacrylamide gel analysis of proteins in mc+ and mc- strains. Crude extracts of exponential cultures of BL322 and BL321 were dialysed overnight against Tris 10 mM pH 7.5, EDTA 1 mM, (-mercaptoethanol 10 mM. The same amounts of proteins of mnc+ and mc- strains were analysed in a 12.5% SDS polyacrylamide gel run under 90V for 18 h. The gel was fixed in 10% acetic acid and stained with Coomassie blue. Lanes 1 and 3: mc+ strain extract (31 and 123 Ag). Lanes 2 and 4: mc- strain extract (30 and 126 yg). The arrows indicate position of the polynucleotide phosphorylase (pnp) and the 1313' subunits of the RNA polymerase (polym.). Other overexpressed proteins are shown by arrow heads.
or lowers translational efficiency, thus allowing a progressive degradation of this mRNA. The importance of RNase III processing of mRNA in the E. coli cell is demonstrated by the numerous differences in the expression of proteins between rnc+ and mc- strains (Figure 4; Gitelman and Apirion, 1980). However, the involvement of RNase III in mRNA stability, long suspected, was never clearly
RNase HI in mRNA decay established (Talkad et al., 1978). In fact, this is the first time to our knowledge that such a strong stabilization of a fully active mRNA is observed as the result of a single mutation. Moreover, it is also the first time that some overproduced proteins are identified. The fact that the proteins identified are polynucleotide phosphorylase and the ,3j' subunits of RNA polymerase can be related to the similarity of the structural organization of the operons which encodes these proteins (Figure 1). Genes for ribosomal proteins are found at the 5' extremities of both operons and are followed by genes involved in RNA metabolism. In addition, there are considerable similarities in their pattern of regulation: L10-L12 are translationally autocontrolled (Fukuda, 1980) as is S15 (Portier, unpublished results). Cotranscription of both operons occurs despite the presence of functional intercistronic terminators (attenuators) (Yamamoto and Nomura, 1978; Portier and Regnier, 1984). The intercistronic RNase Illsensitive site plays apparently the same role in the two operons, since in the absence of processing, there is an overproduction of the downstream encoded proteins. This overproduction would appear to be quantitatively greater for pnp than for (3(' subunits, probably because there is a tight autocontrol of the ((' synthesis of RNA polymerase (Meek and Hayward, 1986). An important question is the relevance of these observations for the growth of wild type E. coli. It might be possible that variations in the level of RNase Ill processing in the cell controls the level of polynucleotide phosphorylase. It is conceivable that under certain physiological conditions, a small decrease in the intracellular amount of RNase III produces a significant increase in polynucleotide phosphorylase level. Conditions known to change the level or activity of RNase III have yet to be identified. In conclusion, prokaryotic gene regulation can be placed in two major categories: transcriptional and post-transcriptional. Up to now, in the latter category, there are many well-established examples of translational autocontrol. However, in these cases, alterations of mRNA stability are thought to have no role in the regulation of gene expression and to be only a secondary consequence of translational feedback regulation (Fallon et al., 1979; Singer and Nomura, 1985). The present study suggests that differential mRNA stability can also be involved in differential expression of genes. The RNase HI processing step which triggers mRNA decay as described here for pnp in an obvious example. Moreover, contrary to the current idea that there are no specific enzymes or structures involved in the functional decay of E. coli mRNA, a RNase HI processing site is an example of a consensus structure which can provide a common target near the extremities of E. coli messages for specific attack by appropriate endonucleases. This can be a clue to unravel the mRNA decay mechanism and also to deepen our knowledge of post-transcriptional control in gene expression.
Materials and methods Products and strains LB medium (Miller, 1972) was made with biotrypcase and yeast extract purchased from BioMerieux (Charbonnieres les Bains, France); MOPS, Tricine and UDP were obtained from Sigma (USA); rifampicin and nalidixic acid were from Ciba-Geigy (Switzerland) and [3H]uridine (30 Ci/mmol), 32p (10 mCi/ml) [ca-32P]CTP (800 Ci/mmol), ['4C]threonine (226 izCi/mmol), were from Amersham (UK). The mc strain together with the isogenic wild type were a gift from W.Studier (1975) to M.Uzan of our laboratory. Relevant characters of the strains used are the following: BL 322: thi-J, argH-1 supE44, BL 321: thi-J, argH-1, supE44, mcl05, JMO1I: Alac, pro, supE, thi (F' proAB, laclq, zAMJ5). Phages M13mp8, Ml3mpl8 and M13mpl9 (and derivatives) were cultured in 2YT medium (Messing, 1983).
Probes The pnp probe used in Northern blot analysis was a 1.7 kb SacHl-MluI fragment cut by restriction endonuclease from plasmid pBPA61 (Portier et al., 1981) and eluted from agarose gel as described (R6gnier and Portier, 1986). The thrS probe used in these experiments was a 1 kb BstEH -SnaI DNA fragment covering almost exclusively the 5' terminal coding sequence (Mayaux et al., 1983). Single-stranded probes for mRNA measurements were constructed by inserting in M13mpl8, M13mpl9 or M13mp8, a fragment of the coding strand of each gene used. The rpsO probe is carried by M13mpl8 and contains a 179 bp fragment extending from the PstI site in the middle of rpsO to the BglI site in the terminator. The pnp probe (1295 bp) corresponds to the central part of the pnp gene, from the SmiaI restriction site to the first PstI site and is cloned in M13mpl9. the thrS probe contains an HpaI fragment of 856 bp inserted in the Ml3mp8 phage and was a gift of M.Springer (Butler et al., 1986). As a control, the same probe with the same fragment cloned in the inverse position was used. M13mpl8 and Ml3mpl9 were used as other controls for hybridization experiments. Labeling and extraction of RNA Rate of synthesis. Strains BL322 and BL321 were grown at 30°C in 20 ml MOPS medium (MOPS 40 mM pH 7.4, Tricine 4 mM pH 7.4, FeSO4 1.8 mM, NH4CI glucose 4 mg/ml, KH2PO4 10 mM, MgSO4 5 mM, NaCl 50 mM, Bl 10 1.3 mM, amino acids 0.4 mM) to an optical density of 0.5 at 600 mn. At this time, 2 ml aliquots were incubated with 100 ACi/ml of [3H]uridine for 2 min and then immediately transferred to a boiling waterbath containing 0.2 ml of Tris 500 mM pH 6.9, EDTA 20 mM, SDS 10%. After vortexing, the tube was incubated 3 min, then 0.2 ml of sodium acetate 2 M pH 5.2 were added, followed by 2 ml of hot phenol. After deproteinization, the sample was centrifuged at 10 000 g for 10 min and the upper phase saved. Another extraction was made with 1.5 ml phenol and 1.5 ml chloroform. The RNA was precipitated with 8 ml alcohol at -20°C overnight. Centrifugation at 10 000 g for 45 min gave a pellet which was digested with 3 Ag/tube of iodoacetate-treated DNase (Zimmerman and Sandeen, 1966). DNase was then inactivated by phenol chloroform extraction and the RNA alcohol-precipitated, rinsed and dried. The pellet was dissolved in 200 Al H2O and radioactivity of an aliquot measured. Decay-rate of mRNA. The cultures (12 ml) were grown and pulse-labelled for 1 min with 100 ACi/ml of [3H]uridine as above, at which point rifampicin (600 Cold uridine (500 1tg/ml) was jig/ml) and nalidixic acid (20 jg/ml) were added. times indicated, 2 ml samples then added at the point taken as 'zero' time. At were withdrawn and incubated at 100°C as described above. After deproteinization, 10 Al of the supernatant was precipitated by 20% trichloroacetic acid, filtered on Millipore filter (type HA 45) and counted to estimate the total incorporation in each fraction. The amount of RNA for each time of incubation was measured again after total purification and the amount of any loss during extraction corrected for. DNA -RNA hybridization Single-stranded DNA probes were prepared as previously described (Messing, 1983) from the different M13 recombinants grown on JM1I1 in 2YT medium at 37°C. The concentration of each probe was measured and equivalent amounts of DNA were fixed on nitrocellulose (Millipore filter type HA 45) by slow filtration and baking at 80°C for 2 h under vacuum. Each filter was loaded with approximately 4 of DNA which corresponds to about 830 fmoles of rpsO DNA, 703 fmoles of pnp DNA and 743 fmoles of thrS DNA. Control filters containing equivalent amount of M13 DNA were used to monitor non-specific hybridization. Each RNA was incubated with at least two separate hybridization series with 0.5 ml hybridization solution: 2 x SSC, tRNA: 100 jig/ml, SDS 0.01 %, pH 7.0 at 66°C for 16 h with gentle agitation. The filters were removed, rinsed, 10 of RNase at 30°C for 60 min. After washed four times and treated with Ig/ml three washings, filters were dried and counted. The radioactivity in specific RNA-DNA hybrids was determined by subtracting the radioactivity associated with control filters. Controls showed that DNA was in large excess and that DNA-RNA hybrids are proportional to the RNA input. Northern blots E. coli BL321 (mc-) and BL322 (mc+) were grown on LB medium at 37°C. Total RNA from exponentially growing cells was isolated by the hot phenol method (Salser et al., 1967). RNA analysis was performed by fractionation on 1 % agarose formaldehyde gels (Maniatis et al., 1982) followed by transfer to an Amersham Hybond-N membrane and hybridization with 32P-labeled DNA fragments (about 108 Cerenkov c.p.m./Ag) as described in the Amersham instruction manual for Hybond-N paper. Double-stranded DNA probes were 32P-labeled by nicktranslation. Relative amounts of mRNA were deduced from densitometric analysis of autoradiographs. When amounts of pnp transcripts were to be determined, 10-fold excess of rnc+ total RNA (12.5-112 itg) over mc- RNA (1. 15-22.3 Ag) was were obtained on the autoradiographs. analysed so that spots of equivalent intensities The ratio of pnp RNA concentrations in mc- strain relative to mc+ strain is the average value of four independent analyses which gave a ratio of 11 + 4.
jtg/ml,
tig
2169
C.Portier et al. Enzyme assays and protein concentrations Exponential cultures of BL322 and BL321 strains were collected at an A6= 0.5, sonicated four times for 15 s and clarified by centrifugation. After overnight dialysis against 0.1 M Tris pH 8.0, 0.4 M NaCl, 0.01 M mercaptoethanol, protein concentration was determined by the Biuret reaction. Polynucleotide phosphorylase and threonyl-tRNA synthetase activities were assayed as described previously (Portier et al., 1981; Plumbridge and Springer, 1982). One unit of polynucleotide phosphorylase corresponds to one itmole of UDP exchanged per mg and per hour. One unit of threonine-tRNA synthetase is defined as one pmole of threonine charged to tRNA per mg and per minute.
Singer,R.P. and Nomura,M. (1985) Mol. Gen. Genet., 199, 543-546. Springer,M., Plumbridge,J.A., Bulter,J.S., Graffe,M., Dondon,J., Mayaux,J.F., Fayat,G., Lestienne,P., Blanquet,S. and Grunberg-Manago,M. (1985) J. Mol. Biol., 185, 93-104. Studier,F.W. (1975) J. Bacteriol., 124, 307-316. Takata,R., Mukai,T. and Hori,K. (1985) Nucleic Acids Res., 13, 7289-7297. Talkad,V., Achord,D. and Kennell,D. (1978) J. Bacteriol., 135, 528-541. von Gabain,A., Belasco,J.G., Schottel,J.L., Chang,A.C.Y. and Cohen,S.N. (1983) Proc. Natl. Acad. Sci. USA, 80, 653-657. Yamamoto,M. and Nomura,M. (1978) Proc. Natl. Acad. Sci. USA, 75,
Acknowledgements
Zimmerman,S.B. and Sandeen,G. (1966) Anal. Biochem., 14, 269-277.
We are grateful to M.Springer for providing thrS probes, to M.Uzan for stimulating discussions, to E.Brody and J.Plumbridge for very careful reading of the manuscript and L.Paineau for typing it. We thank also J.Leautey for helpful assistance in the preparation of double-stranded DNA fragments. P.R6gnier is a fellow of University Paris VII. This work was supported by grants from the Centre National de la Recherche Scientifique: ATP CNRS no. 960141 (to C.P.) and UA 1139 (to M.G.-M.), from the Institut de la Recherche Medicale et de la Sante (Contrat de Recherche Libre no. 831013), from the Fondation pour la Recherche M6dicale and E.I. du Pont de Nemours and Company (to M.G.-M.).
Received on March 30, 1987
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