©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Effects of the Antiterminator BoxA on Transcription Elongation Kinetics and ppGpp Inhibition of Transcription Elongation in Escherichia coli(*)

(Received for publication, March 9, 1995; and in revised form, May 12, 1995)

Ulla Vogel (1) Kaj Frank Jensen (2)

From the  (1)Department of Biological Chemistry and (2)Center for Enzyme Research, Institute of Molecular Biology, University of Copenhagen, S83H, DK-1307 Copenhagen K, Denmark

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

It has been shown previously that two different mRNA chains (lacZ and infB) are elongated at a rate of 40 nucleotides (nt)/s during steady state growth on minimal medium and that the rate of mRNA chain elongation is inhibited by ppGpp in vivo. On the other hand, it was found that a truncated ribosomal RNA chain was elongated at a rate of 80 nt/s, independent of growth condition (Vogel, U., and Jensen, K. F. (1994) J. Biol. Chem. 269, 16236-16241). We reasoned that the different transcriptional behavior of mRNA genes and rRNA operons might be caused by the antiterminator sequences present in the rRNA operons. To test this possibility, we have (a) inserted the minimal antiterminator boxA sequence between the promoter and the lacZ and infB genes and (b) deleted the antiterminator sequences from the rRNA transcription unit and measured transcription elongation rates in vivo on the resulting hybrid genes. We found that insertion of boxA in front of the coding region of lacZ increased the transcription elongation rate from 42 nt/s to 69 nt/s during steady state growth and that it eliminated the ppGpp-dependent decrease in the transcription elongation rate during the stringent response. On the other hand, deletion of the antiterminator sequences from the rRNA operon resulted in a reduced transcription elongation rate, but the elongation rate was still insensitive to changes in the ppGpp pool. These results are consistent with the hypothesis that the antiterminator boxA is a primary determinant of the rate of transcription elongation rate.


INTRODUCTION

When Escherichia coli is exposed to amino acid starvation or other nutritional down-shifts, it undergoes the so-called stringent response, meaning that the synthesis of stable RNA (i.e. tRNA and rRNA) stops abruptly, while bulk mRNA synthesis continues (Cashel and Rudd, 1987), although at reduced rate (Svitil et al., 1993; Set al., 1994). Very early in the stringent response, the unusual nucleotide guanosine 3`,5`-bis(diphosphate), also called ppGpp, accumulates at high concentrations in the cells and is responsible, somehow, for the selective stop of stable RNA synthesis, since relA mutants unable to synthesize high concentrations of ppGpp fail to undergo the stringent response (Cashel and Gallant, 1969; Cashel and Rudd, 1987; Hazeltine and Block, 1973; Pedersen et al., 1973; Stent and Brenner, 1961).

Despite much research, the mechanism by which accumulation of ppGpp leads to down-regulation of tRNA and rRNA synthesis is unknown. Bremer and collaborators (Baracchini and Bremer, 1988; Bremer and Dennis, 1987; Tedin and Bremer, 1992) have suggested that the binding of ppGpp to RNA polymerase leads to partitioning of the enzyme in two forms with different promoter specificity and that the RNA polymerase bound with ppGpp is unable to initiate transcription at stable RNA promoters. On the other hand, Jensen and Pedersen(1990) have proposed that ppGpp inhibits stable RNA synthesis indirectly by inhibiting RNA chain elongation and thereby sequestering RNA polymerase in the elongation phase. Thus, ppGpp was proposed to lower the concentration of free RNA polymerase and reduce the initiation of transcription, primarily from the ribosomal RNA promoters because these strong promoters were considered to be difficult to saturate with RNA polymerase because of unusually high k/K values (Jensen and Pedersen, 1990).

To test the effects of ppGpp on RNA chain growth in vivo, we previously measured the rate of transcription elongation on different genes under different growth conditions of E. coli. We found that the rate of transcription elongation on two different mRNA genes (i.e.lacZ and infB) was about 40 nt(^1)/s during exponential growth on a glucose-salt minimal medium and that it varied slightly with the growth rate (Vogel and Jensen, 1994b). The elongation rate of these mRNAs was approximately halved during the stringent response to isoleucine starvation, when ppGpp accumulated in vast amounts, and it increased to 50 nt/s during the relaxed response to isoleucine starvation, when the basal ppGpp pool decreased to almost undetectable levels (Vogel and Jensen, 1994a; Vogel et al., 1992). These results indicated strongly that mRNA chain elongation is subject to ppGpp inhibition in vivo, since the changes in the elongation rates occurred within the first minute after the onset of isoleucine starvation (Vogel et al., 1992). However, the elongation rate of a truncated ribosomal RNA was close to 80 nt/s, both during steady state growth (in agreement with some previous estimates for intact rRNA (Condon et al., 1993; Molin, 1976) and during amino acid starvation of relA and rel strains, indicating that ppGpp does not inhibit ribosomal RNA chain elongation in vivo.

One difference between the employed mRNA genes and the ribosomal operons is that the latter contain antiterminator sequences similar to the nut sites of bacteriophage (Aksoy et al., 1984; Li et al., 1984) Each ribosomal operon contains two antiterminator sequences, one sequence is placed just after the P2 promoter and the other is positioned in the spacer region between the 16 and 23 S RNA cistrons (Berg et al., 1989). The antiterminators contain three recognizable RNA sequence motifs, namely boxA, boxB, and boxC, but the boxA sequence was shown to be both necessary and sufficient for proper antitermination in the ribosomal operons in vivo and in vitro (Berg et al., 1989; Squires et al., 1993). They are expected to cause binding of different protein factors to the transcription elongation complex (Squires et al., 1993) as shown for the nut sites of bacteriophage (Horwitz et al., 1989), but the binding of such factors to the ribosomal antitermination sites has not been shown.

The experiments reported in this paper were aimed at clarifying the mechanistic basis for the different responses to ppGpp pools variations between transcription complexes on ribosomal RNA operons and mRNA genes. First, we have studied the rate of transcription elongation in a DeltarelA DeltaspoT strain (i.e. a ppGpp^o mutant devoid of ppGpp) to observe the effects of basal level ppGpp pools without disturbing the growth. Second, we have studied the effects on transcription elongation of inserting the antiterminator boxA in front of the coding regions of infB and lacZ transcripts and of removal of the antiterminator boxes from a ribosomal operon. The results show conclusively that the antiterminator boxA is a major determinant of both transcription elongation rate and of resistance to ppGpp pool changes in vivo.


EXPERIMENTAL PROCEDURES

Strains and Plasmids

Unless stated otherwise, the background strain used for these experiments is MAS90, i.e.E. coli K-12 thi Deltapro-lac relA2/F`lacIlacZ::Tn5 proAB, which has been described previously (Vogel and Jensen, 1994a). CF1693 (MG1655 DeltarelA251 DeltaspoT207) and CF1648 (MG1655) (Xiao et al., 1991) were gifts from V. J. Hernandez (National Institutes of Health, Bethesda, MD). The F` proABlacIlacZ DeltaM15 Tn10 from SØ6645 (MC1061, pyrD::Km/F` proABlacIlacZ DeltaM15 Tn10) (own collection), which confers tetracycline resistance, was mated into the two strains selecting tetracycline resistance and pyrimidine prototrophy. The transconjugants were subsequently transformed with either pUV12 or pUV16 selecting for ampicillin resistance. The construction of the plasmids pUV12, pUV14, and pUV17 is described elsewhere (Vogel and Jensen, 1994a).

Construction of pUV22

An EcoRI-EcoRI fragment from pUV17 containing the minimal boxA and 2122-bp rDNA was exchanged with an EcoRI-EcoRI fragment from pKK3535 (Brosius et al., 1981) containing the same 2122-bp sequence and 30 additional bp in the 5` end of the rDNA fragment, but not containing the minimal boxA.

Construction of pUV26

The antitermination sequences in the spacer region of the rDNA in pUV22 were removed by PCR amplifying a segment of rDNA on either side of the antiterminator sequences, incorporating a BamHI site into the primers. The 5` PCR fragment had an XmaI site in the rDNA sequence in the 5` end and a BamHI site in the 3` end. The 3` PCR fragment had a BamHI site in the 5` end and a BssHII site in the rDNA sequence at the 3` end. The two PCR fragments were digested with the appropriate restriction enzymes and ligated into XmaI-BssHII-digested pUV22 in a three-fragment cloning resulting in deletion of 84-bp rDNA and insertion of 4 bp from the artificial BamHI site.

Construction of pUV16

pUHE23-2 is a cloning vector containing the synthetic, IPTG-inducible promoter P, which is a derivative of the T7 A1 promoter. The promoter is fused with two lac operator sites, followed by a polylinker, a terminator structure, a cat gene, and the ribosomal T1 terminator. The plasmid confers ampicillin resistance (H. Bujard, University of Heidelberg, Germany). A DNA duplex made by hybridization of the two synthetic oligonucleotides, 5`-AATTCTAGACACTGCTCTT-TAACAATTTAG-3` and 5`-GATCCTAAATTGTTAAAGAGCAGTGTCTAG-3` containing the minimal boxA sequence (Squires et al., 1993) and flanking sequences was digested with EcoRI and BamHI and inserted into EcoRI-BamHI-digested pUHE23-2. The operation resulted in the deletion of 33 nucleotide residues from the polylinker region, containing an unused Shine-Dalgarno sequence, and the insertion of 30 nucleotide residues. The resulting plasmid is called pUV15. pUV15 was digested with BamHI and PvuII and a 3,100-bp BamHI-DraI fragment containing the entire lacZ reading frame from pTL25 (Linn and Ralling, 1985) was inserted behind the boxA sequence.

Construction of pUV25

pUV15 was digested with BamHI and PvuII, cutting just behind the boxA sequence and in the cat gene, respectively. A 3,111-bp BglII-NruI fragment from pA2-1 (Plumbridge and Springer, 1983) containing the entire infB and a small part of the 5` part of the 15K gene was inserted behind the boxA sequence, resulting in an in-frame fusion of 15K` and `cat.

Growth Conditions

The bacteria were grown in A + B medium (Clark and Maal, 1967) supplemented with thiamine (1 µg/ml), ampicillin (100 µg/ml), and glucose (0.2%). CF1648 and CF1693 derivatives were also supplemented with casamino acids (0.2%), uracil (20 µg/ml), and tetracycline (30 µg/ml). The bacteria were grown at 37 °C. Isoleucine starvation was induced by addition of 0.4 mg/ml valine (Leavitt and Umbarger, 1962). Transcription from P was induced by 1 mM IPTG.

Induction Lag for beta-Galactosidase Synthesis

Sampling was performed as described by Vogel and Jensen (1994b), beta-galactosidase activities were determined as described by Miller(1972), and the data were analyzed in the square root plot of Schleif et al. (1973).

RNA Techniques

RNA sampling and purification was performed as described previously (Vogel et al., 1992); dot blots and hybridization were performed as described by Vogel and Jensen (1994a). The dot blots were quantified on an Instant Imager from Canberra-Packard and subjected to autoradiography. The hybridized radioactivity (cpm) was depicted as a function of time to give an induction curve. When the curves were fitted, the earlier time points were weighted more heavily than the later time points due to instability of the transcripts (Vogel and Jensen, 1994a). For control purposes, parallel dot blots were made using a probe complementary to the bla transcript in each experiments. In vitro transcription of riboprobe plasmids with [alpha-P]UTP (DuPont NEN) and plasmids used for the in vitro transcription have been described previously (Vogel and Jensen, 1994a; Vogel et al., 1992).


RESULTS

There are at least two straightforward explanations for the faster transcription elongation on rRNA genes compared to mRNA genes. 1) The rRNA sequences may lack ppGpp-dependent pause sites, presumed to make RNA polymerase elongate mRNA chains more slowly when the ppGpp pool increases. 2) The antiterminator boxes and the putative factors, which may bind to RNA polymerase at the boxes, might make the polymerase resistant to ppGpp-dependent (and ppGpp-independent) pausing, thereby increasing the over-all transcription elongation rate. To distinguish between these non-excluding alternatives, we have studied the effects on transcription elongation of removing the antiterminator boxes from a ribosomal operon and of inserting an antiterminator boxA sequence in the early transcribed regions of infB and lacZ.

Measurements of the rates of transcription elongation were made according to a previously published procedure (Vogel et al., 1992) using the plasmids described in Fig. 1. Thus, the time lag between induction of a plasmid-borne, lacI repressor-controlled promoter and detection of specific hybridization to a probe complementary to the 3` end of the transcript was measured and taken to represent the transcription time of the gene. The promoter on these plasmids is a modified T7 A1 promoter, the P of plasmid pUHE23-2, and it was kept repressed, until induction, by a lacI^q present on an episome. Transcription was induced at time zero by addition of IPTG, and samples were withdrawn every 10 s for RNA purification. The RNA samples were analyzed by dot blot hybridization to the appropriate riboprobes shown in Fig. 1, and the amount of radioactive riboprobe hybridized to each RNA sample was quantified on an Instant Imager (Canberra-Packard). Autoradiograms of two sets of dot blots are shown in Fig. 2.


Figure 1: Schematic presentation of the employed plasmids. Genes are represented by openbars. P/O, promoter/operator site of P; T, transcription terminator sequence; `, truncated gene; thicklines, probe complementary to the transcripts at the indicated positions relative to the 5` end; boxA, the minimal antiterminator boxA (in the 5` end of the transcripts); AT, the natural antiterminator sequences in the spacer region of the rDNA in plasmids pUV17 and pUV22.




Figure 2: Autoradiograms of dot blots from induction experiments using MAS90 harboring pUV25. Induction was performed during growth on glucose minimal medium. Top, steady state growth; bottom, isoleucine starvation induced by addition of valine (0.4 mg/ml) 6 min prior to induction of transcription with IPTG (1 mM). The first sample is the uninduced background, followed by samples are taken out every 10 s from 20 to 120 s.



lacZ mRNA Chain Elongation with and without BoxA

Plasmid pUV12 contains the normal lacZ gene transcribed from the P promoter. In pUV16, the minimal boxA sequence was inserted between the P promoter and the structural lacZ gene. Fig. 3shows the kinetics of appearance of full-length lacZ transcripts from these two plasmids following induction with IPTG. During steady state growth, the transcription time was 75 s for the normal lacZ gene, corresponding to an elongation rate of 41 nt/s. When boxA was inserted after the promoter, transcription elongation occurred considerably faster as the transcription time was only 46 s, corresponding to 67 nt/s. This difference in the transcription kinetics for the lacZ gene, in the presence and absence of boxA, was even more pronounced during the stringent response (Fig. 3, panelsC and D) since the transcription times for the lacZ mRNA during the stringent response were 50 s (= 62 nt/s) in the presence of boxA (pUV16) and 160 s (= 19 nt/s) without the antiterminator boxA (pUV12). These results indicate that boxA is a primary determinant of the rate of transcription elongation and of the lack of sensitivity to ppGpp accumulation during the stringent response.


Figure 3: Induction of lacZ mRNA synthesis in MAS90 harboring either pUV12 (A and C) or pUV16 (B and D). MAS90 was grown on glucose minimal medium. At time = 0, IPTG was added at 1 mM. Transcription was either induced during steady state growth (A and B) or 6 min after the onset of isoleucine starvation induced by addition of 0.4 mg/ml valine (C and D). RNA was extracted at the indicated times after induction and used for dot blot hybridization with the 3`-lac probe (see Fig. 1). The amount of hybridization was quantified on an Instant Imager (Canberra-Packard) and cpm was plotted versus sampling time. All the experiments were performed at least two times.



Effects of boxA on Transcription Polarity

Fig. 4shows induction experiments with pUV12 and pUV16, where a probe complementary to the 5` part of the lacZ transcript (see Fig. 1) was used. The slopes of the induction curves, monitored with this early probe, reflect the relative initiation rates and differences in the polarity in the very early part of the gene. There is no difference between the slopes of the induction curves of the boxA-lacZ transcript (Fig. 4B) during steady state growth and during the stringent response, indicating that the initiation rate of the T7 A1 promoter is unchanged during the stringent response in agreement with earlier observations that initiation of transcription at the T7 A1 promoter is resistant to ppGpp in vitro (Kingston et al., 1981). As the full-length boxA-lacZ transcript also accumulates at similar rates during exponential growth and amino acid starvation (Fig. 3, panelsB and D), it appears that amino acid starvation does not induce transcription polarity in lacZ when the boxA sequence is contained in the mRNA. On the other hand, for the normal lacZ gene on pUV12, it is evident that amino acid starvation causes a strong transcriptional polarity, since the slopes of the induction curves probed with the 5`-probe deviate from each other by 30% (Fig. 4A), while the slopes of the induction curves probed with the 3`-probe deviate from each other by a factor of 9 (Fig. 3, panelsA and C).


Figure 4: Induction of the 5`-lacZ mRNA in MAS90 carrying either pUV12 (A) or pUV16 (B) using the 5`-lacZ probe (see Fig. 1). Transcription was either induced during steady state growth (filledcircles) or 6 min after the onset of isoleucine starvation induced by addition of 0.4 mg/ml valine (opencircles). The RNA samples were the same as those used for Fig. 3, but the 5`-lac probe (Fig. 1) was used for quantitative hybridization. The experiments with pUV12 were made twice.



Effects of BoxA on infB mRNA

In order to ensure that the effects of boxA on mRNA chain elongation were not specific for the lacZ gene, we also inserted the antiterminator boxA in front of infB (plasmid pUV25). The induction kinetics of the antiterminated infB transcript on plasmid pUV25 are shown in Fig. 5. The results are qualitatively similar to the results obtained for the antiterminated lacZ gene, since the transcription elongation rate (70 nt/s) for the antiterminated infB gene during exponential growth was higher than for the infB gene without boxA, i.e. 42 nt/s (Vogel and Jensen, 1994a) and since it remained high during the stringent response.


Figure 5: Induction of infB mRNA synthesis in MAS90 harboring pUV25. Transcription is induced either during steady state growth (A) or 6 min after the onset of isoleucine starvation induced by the addition of 0.4 mg/ml valine (B). The experimental procedures were the same as those for Fig. 3.



Transcription Elongation Rates in a ppGpp^oStrain

We observed previously that the rate of transcription elongation increased slightly during the relaxed response to isoleucine starvation, when the basal ppGpp pool decreased to almost undetectable levels (Vogel and Jensen, 1994a). This observation indicated that even basal level ppGpp pools inhibit mRNA chain elongation. Therefore, we measured the elongation rate on the normal lacZ gene (on plasmid pUV12) and on the antiterminated lacZ gene (on plasmid pUV16) in the ppGpp^o mutant CF1693 and the parental strain (Xiao et al., 1991) MG1655 (CF1648). These strains contain a functional chromosomal lac operon, but mRNA synthesis from the chromosomal lacZ gene was ignored, since dot blots of the later time points after induction of plasmid free cells only rose by 30 cpm (results not shown), which constitutes less than 1.5% of the amount of hybridization seen with the plasmid carrying cells.

Fig. 6shows the transcription kinetics from lacZ (pUV12) and from the antiterminated lacZ (pUV16) in wild type MG1655 (CF1648) and in the DeltarelA DeltaspoT derivative (CF1693). The transcription time of the antiterminated lacZ transcript did not vary significantly between the two strains as the 5-s difference observed between the two strains is on the limit of detection in our assay, but the transcription time of the normal lacZ, gene was 60 s in the DeltarelA DeltaspoT strain and 80 s in the wild type. These results confirm that even basal level ppGpp concentrations inhibit mRNA transcription elongation.


Figure 6: Induction experiments using CF1648 relA spoT (A and B) or CF1693 DeltarelA DeltaspoT (C and D) harboring either pUV12 (A and C) or pUV16 (B and D). The cells were grown in glucose minimal medium supplemented with casamino acids and uracil. The experimental procedures were the same as those for Fig. 3. The experiments with the DeltarelA DeltaspoT strain were made twice, while the experiments with the relAspoT strain only were performed once in this background strain.



Effects of a High mRNA Chain Elongation Rate on Translation

As shown in Fig. 7, the presence of the antiterminator boxA sequence on lacZ transcript did not affect the induction lag of beta-galactosidase, indicating that the rate of translation is not increased when the rate of transcription is increased. This result is at variance with the result of Jin et al.(1992), who observed a 10% reduced induction lag for beta-galactosidase in cells harboring the rpoB3595 mutation that makes RNA polymerase transcribe faster. The difference may be due to strain differences, but our observation is in accordance with the finding that RNA polymerase does not seem to limit ribosome movement during steady state growth (Set al., 1994; Vind et al., 1993).


Figure 7: Square root plot of the increment in beta-galactosidase activity over the uninduced level in MAS90. Filledcircles, pUV12; opencircles, pUV16. MAS90 was grown in glucose minimal medium. At time zero, beta-galactosidase synthesis was induced by addition of 1 mM IPTG. Samples were pipetted into chloramphenicol at the indicated times and the activity determined as described by Miller(1972).



Transcription of rRNA with and without BoxA

The plasmids described below and in Fig. 1contain different combinations of a promoter proximal boxA sequence and the boxA sequence in the spacer region of the ribosomal rrnX operon. In pUV17, the P promoter is fused to an artificial minimal antiterminator boxA preceding a segment of rDNA and the 3` part of the cat gene. The rDNA contains the 3` part of the 16 S region, the spacer region, and the 5` part of the 23 S region, including the internal antiterminator sequence. In pUV22, the minimal antiterminator box in front of the rDNA segment was replaced by rDNA, and in pUV26 also the internal antiterminator sequence was removed by deleting 80 bp from the spacer region.

As shown in Fig. 8(panels A-C), removal of the antitermination boxes led to a successive increase in the transcription time during steady state growth, i.e. from 45 s for pUV17, to 55 s for pUV22 and 70 s for pUV26. These numbers correspond to transcription elongation rates of 80 nt/s in the presence of both antiterminator boxes (pUV17), a transcription elongation rate of 65 nt/s when only the antiterminator in the spacer region is present (pUV22) and 50 nt/s without any of the two antiterminators (pUV26), but the transcription elongation rate even without any antiterminator boxes is still significantly higher than we observe for the mRNAs, i.e. 40 nt/s (see below). Moreover, as seen from Fig. 8(panels D-F), the transcription elongation rate from the modified rrnX operon on any of these plasmids was not reduced during the stringent response as normally seen for mRNA, indicating either that antiterminator sequences do not influence ppGpp inhibition or, simply, that the transcribed rDNA segments in the plasmids do not contain ppGpp specific pause sites.


Figure 8: Induction of truncated rRNA in MAS90 carrying either pUV17 (A and D), pUV22 (B and E), or pUV26 (C and F). MAS90 was grown on glucose minimal medium. At time = 0, IPTG was added at 1 mM. Transcription was either induced during steady state growth (A-C) or 6 min after the onset of isoleucine starvation induced by addition of 0.4 mg/ml valine (D-F). RNA was extracted at the indicated times after induction and used for dot blot hybridization with the cat probe. The amount of hybridization was quantified on in an Instant Imager (Canberra-Packard) and cpm was plotted versus sampling time. All the experiments were performed twice. The data in panelD were taken from (Vogel and Jensen, 1994a) and are incorporated in the figure for clarity reasons.




DISCUSSION

Our results demonstrate that the boxA sequence, in addition to the well established role in antitermination, also functions as a determinant of the rate of transcription elongation. Moreover, it appears that the presence of an antiterminator boxA early after the promoter makes transcription elongation insensitive to inhibition by ppGpp. The results of all transcription experiments are summarized in Table 1. It is clear that boxA divides the data in two groups on the basis of elongation rates, since the RNA chains that carry the boxA sequence are elongated at high rates (62-82 nt/s), while the RNAs that do not have boxA are elongated more slowly (19-52 nt/s). It is also clear that the elongation rate of mRNAs, devoid of the boxA sequence, responds strongly (up to 2.6-fold) to changes in the ppGpp pool, while the elongation rate of the corresponding mRNAs carrying boxA only changes by 25-30% in response to similar changes to the ppGpp pool. These observations indicate that the boxA sequence, or the protein factors that associate with RNA polymerases at boxA, make RNA polymerase more resistant to ppGpp inhibition.



In this context, the results obtained from transcription studies of a segment of rRNA are relevant. These data also support the notion that boxA is a major determinant of the RNA chain elongation rate, since transcription of rRNA in the absence of antitermination sequences occurred with an elongation rate of 50 or 80 nt/s in the presence of antiterminator sequences. However, even after deletion of the antiterminator boxes, the elongation rate of the truncated rrnX operon DNA was still higher than observed for the mRNAs lacZ and infB, and it did not respond significantly to ppGpp accumulation during the stringent response. That observation may indicate that the rDNA segment on these plasmids is devoid of ppGpp-dependent pause sites for transcription. The absence of such transcriptional pauses would also explain why the elongation rate of rRNA lacking antiterminator sequences (50 nt/s) is equal to the elongation rate of the lacZ mRNA in the ppGpp^o strain, but higher than the elongation rate (42 nt/s) of the lacZ mRNA during steady state growth in the presence of the basal level ppGpp pool.

Under conditions of isoleucine starvation, we see about 80-90% transcription polarity in lacZ, and that polarity is completely suppressed in the boxA-lacZ fusion. Also, the data in Table 1indicate the existence of a correlation between a low rate of transcription elongation and a high degree of transcriptional polarity, since the stringent response induced 80-90% polarity in the lacZ gene (see text), 50% polarity in the infB gene (Vogel and Jensen, 1994a), and no (or extremely little) polarity in the antiterminator-free rrn-cat transcript (Fig. 8, panelsC and F). We therefore considered the possibility that the observed changes in the elongation rate, in response to changes in growth condition, were secondary results of the changes in transcription polarity (or vice versa, that the observed levels of polarity were secondary results of the RNA chain elongation rates). However, we ruled out that suggestion because the transcription elongation rate on lacZ decreased during the stringent response to isoleucine starvation and increased during the relaxed response, while the transcriptional polarity was higher during the relaxed response than during the stringent response (Vogel and Jensen, 1994a).

Somehow, we find it puzzling that rRNA transcription elongation is insensitive to ppGpp, since the down-regulation of ribosomal RNA synthesis is known as a primary target for ppGpp action during the stringent response (Cashel and Rudd, 1987). On the other hand, since the high concentration of ppGpp accumulating under the stringent response inhibits mRNA elongation, ppGpp will serve to minimize decoupling of the ribosomes from the RNA polymerase, and thereby reduce the level of premature transcription termination, under growth conditions that give rise to suboptimal ribosome function. Furthermore, high concentrations of ppGpp lead to reduction of the total mRNA pool; thus, indirectly, ppGpp enhances the accuracy of translation during shortage of substrates for the elongating ribosomes (Svitil et al., 1993; Set al., 1993, 1994). It appears as if the `naked` RNA polymerase is designed to elongate slowly enough to allow ribosomes to catch up and to be inhibited to ppGpp, which is a signal for ribosome starvation. However, the RNA polymerase clearly has the capacity to elongate RNA chains much faster, as evidenced by the high elongation rate of ribosomal RNA. The function of the high transcription elongation rate of rRNA may be to make the polymerase less prone to terminate transcription, as proposed for Rho-dependent termination on the basis of analysis of RNA polymerase mutants (Jin et al., 1992), and to reduce the number of RNA polymerases needed for rRNA transcription and thus for total transcription.

To conclude, the antiterminator boxes seem to have three functions: 1) they prevent polarity, 2) they increase the rate of transcription elongation, and 3) they confer ppGpp insensitivity to the RNA polymerase. In turn, these observations indicate that these functions of the RNA polymerase (polarity, elongation rate, and ppGpp sensitivity) are intimately coupled with each other.


FOOTNOTES

*
The Center for Enzyme Research is funded by the Danish National Research Foundation, and the work was also supported by the Danish Center for Microbiology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^1
The abbreviations used are: nt, nucleotide(s); bp, base pair(s); IPTG, isopropyl-1-thio-beta-D-galactopyranoside; PCR, polymerase chain reaction; ppGpp, guanosine 3`,5`-bis(diphosphate).


ACKNOWLEDGEMENTS

We thank Michael S(Institute of Molecular Biology, Copenhagen, Denmark) for strains and plasmids. Furthermore, we thank Catherine Squires (Tufts Medical School, Boston, MA) for advice on the antiterminator boxA, V. J. Hernandez (National Institutes of Health, Bethesda, MD) for the strains CF1648 and CF1693, and Soumaya Laalami (Pasteur Institute, Paris, France) for the generous gift of pA2-1.


REFERENCES

  1. Aksoy, A., Squires, C. L. & Squires, C.(1984)J. Bacteriol. 159,260-264 [Medline] [Order article via Infotrieve]
  2. Baracchini, E. & Bremer, H.(1988)J. Biol. Chem.263,2597-2602 [Abstract/Free Full Text]
  3. Berg, K. L., Squires, C. & Squires, C. L.(1989)J. Mol. Biol. 209,345-358 [Medline] [Order article via Infotrieve]
  4. Bremer, H. & Dennis, P. P. (1987) in Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology (Neidhardt, F. C., Ingraham, J. C., Brooks-Low, K., Magasanik, B., Schaecter, M. & Umbarger, H. E., eds) pp. 1527-1542, American Society for Microbiology, Washington, D. C.
  5. Brosius, J., Dull, T. J., Sleeter, D. D. & Noller, H. F.(1981)J. Mol. Biol.148,107-127 [Medline] [Order article via Infotrieve]
  6. Cashel, M. & Gallant, J.(1969)Nature221,838-841 [Medline] [Order article via Infotrieve]
  7. Cashel, M. & Rudd, K. E. (1987) in Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology (Neidhardt, F. C., Ingraham, J. C., Brooks-Low, K., Magasanik, B., Schaecter, M. & Umbarger, H. E., eds) pp. 1410-1438, American Society for Microbiology, Washington, D. C.
  8. Clark, D. J. & Maal, O.(1967)J. Mol. Biol.23,99-112
  9. Condon, C., French, S., Squires, C. & Squires, C. L.(1993)EMBO J. 12,4305-4315 [Abstract]
  10. Hazeltine, W. A. & Block, R.(1973)Proc. Natl. Acad. Sci. U. S. A. 70,1564-1568 [Abstract]
  11. Horwitz, R. J., Li, J. & Greenblatt, J.(1989)Cell51,631-641
  12. Jensen, K. F. & Pedersen, S.(1990)Microbiol. Rev.54,89-100
  13. Jin, D. J., Burgess, R. R., Richardson, J. P. & Gross, C. A.(1992)Proc. Natl. Acad. Sci. U. S. A.89,1453-1457 [Abstract]
  14. Kingston, R. E., Nierman, W. C. & Chamberlin, M. J.(1981)J. Biol. Chem.256,2787-2797 [Abstract/Free Full Text]
  15. Leavitt, R. I. & Umbarger, H. E.(1962)J. Bacteriol.83,624-630 [Medline] [Order article via Infotrieve]
  16. Li, S., Squires, C. & Squires, C.(1984)Cell38,851-860 [Medline] [Order article via Infotrieve]
  17. Linn, T. & Ralling, G.(1985)Plasmid14,134-142 [Medline] [Order article via Infotrieve]
  18. Miller, J. H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  19. Molin, S. (1976) in Alfred Benzon Symposium IX (Kjeldgaard, N. O. & Maal, O., eds) pp. 333-337, Academic Press, Inc., New York
  20. Pedersen, F. S., Lund, E. & Kjeldgaard, N. O.(1973)Nat. New Biol. 243,13-15
  21. Plumbridge, J. A. & Springer, M.(1983)J. Mol. Biol.167,227-243 [Medline] [Order article via Infotrieve]
  22. Schleif, R., Hess, W., Finkelstein, S. & Ellis, D.(1973)J. Bacteriol. 115,9-14 [Medline] [Order article via Infotrieve]
  23. S, M. A., Vogel, U., Jensen, K. F. & Pedersen, S.(1993) Antonie Leeuwenhoek63,323-331 [Medline] [Order article via Infotrieve]
  24. S, M. A., Jensen, K. F. & Pedersen, S.(1994)J. Mol. Biol. 236,441-454 [CrossRef][Medline] [Order article via Infotrieve]
  25. Squires, C. L., Greenblatt, J., Li, J., Condon, C. & Squires, C. L.(1993) Proc. Natl. Acad. Sci. U. S. A.90,970-974 [Abstract]
  26. Stent, G. S. & Brenner, S.(1961)Proc. Natl. Acad. Sci. U. S. A. 47,2005-2014 [Medline] [Order article via Infotrieve]
  27. Svitil, A. L., Cashel, M. & Zyskind, J. W.(1993)J. Biol. Chem. 268,2307-2311 [Abstract/Free Full Text]
  28. Tedin, K. & Bremer, H.(1992)J. Biol. Chem.267,2337-2344 [Abstract/Free Full Text]
  29. Vind, J., S, M. A., Rasmussen, M. D. & Pedersen, S. (1993)J. Mol. Biol.231,678-688 [CrossRef][Medline] [Order article via Infotrieve]
  30. Vogel, U. & Jensen, K. F.(1994a)J. Biol. Chem.269,16236-16241 [Abstract/Free Full Text]
  31. Vogel, U. & Jensen, K. F.(1994b)J. Bacteriol.176,2807-281 [Abstract]
  32. Vogel, U., S, M., Pedersen, S., Jensen, K. F. & Kilstrup, M. (1992)Mol. Microbiol.6,2191-2200 [Medline] [Order article via Infotrieve]
  33. Xiao, H., Kalman, M., Ikehara, K., Zemel, K., Glaser, G. & Cashel, M.(1991) J. Biol. Chem.266,5980-5990 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.