Conserved sequence elements upstream and downstream from the transcription initiation site of the Caulobacter crescentus rrnA gene cluster

J. Mol. Biol. (1989) 210, 245-254

Conserved Sequence Elements Upstream and Downstream from the Transcription Initiation Site of the Caulobacter crescentus rrnA Gene Cluster Kei Amemiya National Institutes of Health (NINDS) Laboratory of Viral and Molecular Pathogenesis Bethesda, MD 20892, U.S.A. (Received 13 January

1989, and in revised form

17 July

1989)

The nucleotide sequence and in vivo transcription start sites for rrnA, one of the two rRNA gene clusters of the eubacterium Caulobacter crescentus, have been determined. Two transcription start sites, a major and minor, for the rRNA gene cluster are located more than 700 nucleotides upstream from the 16 S rRNA gene. Transcription was detected from only the major start site in swarmer cells, But after the swarmer-to-stalked cell transition, transcription was detected from both rRNA start sites and continued throughout the developmental cell cycle when cells were grown in minimal medium. On the other hand, transcription from only the major start site was detected in cells growing in a complex medium. A small open reading frame was found upstream from the rRNA gene transcription start sites and was followed by an inverted repeat sequence. No sequence homology was found between the major rRNA gene transcription start site and the Escherichia coli 0” promoters or the consensus sequence elements reported for C. crescentus JEa promoters. However, there were two areas of homology when the major rRNA gene promoter was compared to the nucleotide sequence of the C. crescentus trpFBA promoter. There was a 12 nucleotide sequence centered around the - 10 region of both promoters that was closely homologous. In addition, immediately downstream from the transcription start there was a sequence element that was identical in both promoters. These nucleotide sequence elements were not in the temporally expressedJEa promoters of C. crescentus.

1. Introduction

and a motile swarmer cell. The mature stalk cell begins to elongate, and eventually, forms a new swarmer cell at the pole opposite the stalk. On the other hand, the swarmer ceil begins a series of developmental events that leads to the formation of a new stalk ceil. DNA synthesis appears to be initiated concurrently with the formation of a new stalk structure and continues as the cell matures into a stalk cell and begins the elongation and cell division phase of development. Genes that encode the components of the surface structure apparatus, such as the basal body, hook and flagella, and that regulate their synthesis are being examined to determine the &s-acting nucleotide sequence elements that permit their temporal regulation (Milhausen et al., 1982; Ohta et al., 1982, 1985; Purucker et al., 1982; Gill & Agabian, 1983; Ohta & Newton, 1984; Ohta et aZ., 1985; Chen et al., 1986; Hahnenberger & Shapiro, 1987; Minnich & Newton, 1987; Mullin et al., 1987). In addition to these temporally regulated genes, we have also been studying the ribosomal RNA (rRNA) genes of C. crescentus in order to examine

During the development of a cell a complex program of gene expression is temporally regulated at many different levels. In order to understand how gene expression is regulated at the level of transcription, components in the transcription process must be identified and their interaction with specific cisacting sequence elements characterized. It has become evident that, as more genes are isolated and their regulatory regions elucidated, all promoters do not share common nucleotide sequence elements (Cowing et al., 1985; Helmann & Chamberlin, 1987; Mullin et al., 1987) and that a single form of RNA polymerase is involved in the expression of these genes (Losick C Pero, 1981; Grossman et al., 1984; Hirschman et al., 1985; Hunt & Magasanik, 1985; Fujita et al., 1987). Caulobacter crescentus is a eubacterium that undergoes a series of obligate differentiation events (for reviews, see Newton et al., 1985; Shapiro, 1985). Upon an asymmetric cell division event two distinct cell types are produced: a non-motile stalked cell 245 0022-2836/89/220245-10

$03.00/O

0

1989 Academic

Press Limited

246

K. Amemiya

their structure, expression, and maturation during the C. crescentus cell cycle (Bellofatto et al., 1983; Feingold et al., 1985; Amemiya et al., 1986). It has been determined that C. crescentus has only two copies of rRNA gene clusters and that the organization in each rRNA gene cluster is 5’-16 S-tRNA spacer-23 S-5 S-3’ (Ohta BENewton, 1981; Feingold et al., 1985). Although the region up to 300 nucleotides 5’ to one of the 16 S rRNA genes has been isolated (Ohta & Newton, 1981; Feingold et aZ., 1985) and. the nucleotide sequence of this region determined, the promoter for this gene cluster was not found (Feingold et al., 1985). This conclusion was supported by the absence of specific RNA polymerase binding and transcription initiation sites in the 5’ region of the isolated rRNA gene cluster (Feingold et al., 1985; Amemiya et al., 1986). We now report the nucleotide sequence and in vivo transcription start for sites for one of the rRNA genes from C. crescentus.

2. Materials and Methods (a) Bacterial strains, phages and plasmids C. crescentus CB15 was grown at 30°C in either peptoneyeast extract (PYE) broth (Poindexter, 1964) or in a minimal medium (M2) with @2% (w/v) glucose (Contreras et al., 1978). When required, synchronized cultures of C. crescentus were obtained by a slight modification of the procedure of Evinger 6 Agabian (1977) that entailed the use of Ludox LS (DuPont) instead of Ludox HS. Cells were grown in M2 medium to an optical density of 0% to 1.0 at 660 nm and mixed with cold Ludox LS at a ratio of 1 part Ludox to 3 parts cell culture. After centrifugation at 7500 revs/min for 15 min, the lower layer, consisting of swarmer cells, was removed and washed twice with cold M2 medium without glucose. This procedure resulted in a swarmer population of at least. 90 to 95% purity. Growth was initiated by placing the cells in prewarmed M2 medium with glucose. Escherichia coZi HBlOl was grown at 37°C in L-broth under conditions described by Maniatis et al. (1982). E. coli Q358, which was obtained from N. Ohta (Princeton University), was grown at 37°C in L-broth supplemented with @2 y. (w/v) maltose or in NZC broth (Maniatis et al., 1982). E. coli JMlOl or JM105 was grown as dgscribed (Pharmacia, 1984). The Xharon 4 recombinant Cc5507 (Ohta & Newton, 1981), which contains the $-flanking region of a ribosomal RNA gene from C. crescentus CB15, was kindly supplied by N. Ohta and A. Newton (Princeton University). The single-stranded phages, M13mp18 and M13mp19, were obtained from Pharmacia. The broad host-range plasmid pRK290 (Ditta et aE., 1980) was obtained from B. Ely (University of South Carolina), and pUC19 wm obtained from B. Alexander in our laboratory. (b) Enzymes and reagents Restriction enzymes were obtained from BoehringerMannheim, International Biotechnologies Inc., or New England Biolabs and were used as specified by the manufacturer. S1 nuclease, calf intestine alkaline phosphatase, and phage T4 polynucleotide kinase were obtained from Radioactive nucleotides Boehringer-Mannheim. and [cr-32P]dATP or [1-32P]ATP (10 Ci/mmol)

[a-32P]dCTP Amersham.

(3000 Ci/mmol)

were

obtained

from

(c) DNA sequencing and detection subcloning The dideoxynucleotide method of Sanger et al. (1977) was used to determine the nucleotide sequence of DNA fragments. Deletions in DNA fragments cloned into Ml3 phages were obtained by the method of Dale et al. (1985), and reagents for creating the deletions were obtained from International Biotechnologies Inc. The DNA sequence ladder (purine reactions) for sizing S, nuclease-resistant products was obtained by the method of Maxam & Gilbert ( 1980). (d) S 1 nuclease assay S, nuclease mapping was performed essentially as described by Berk & Sharp (1977) and modified as described (Amemiya et al., 1986). Each assay contained 100 pg of RNA prepared as described (Amemiya et al., 1980) and hybridized with end-labeled DNA fragments at 46°C for 3 to 4 h. Between 2000 and 3000 units of S, nuclease were used for each assay. Samples were analyzed on a 6% polyacrylamide slab gel containing 7 M-urea in 45 mM-Tris.HCl (pH 8.0), 45 mnil-boric acid, 1.25 mMEDTA, and the gels were dried before autoradiography. Densitometer tracings of S, autoradiograms were performed with a Joyce-Loebl MKIIIC microdensitometer. The initiating nucleotide was determined by comparison with the migration of the Maxam & Gilbert (1980) purine reactions of the same DNA probe. Adjustments were made for the difference in migration between the 8, nuclease-resistant product and the purine sequencing lanes (Sollner-Webb & Reeder, 1979).

3. Results (a) Isolation

of the region 5’ to the 16 S rRNA

gene

The region 5’ to the 16 S rRNA gene was isolated from the Xharon 4 clone Cc5507 (Ohta & Newton, 1981). Cc5507 reportedly consisted of five EcoRI restriction fragments that contained the 16 S, spacer tRNA region, 23 S and 5 S rRNA genes. One of the EcoRI fragments, a 42 kbt fragment, was located 5’ to a 3.4 kb EcoRI fragment that contained the entire 16 S rRNA gene, the tRNA spacer region, and part of the 23 S rRNA gene. We had examined the 3.4 kb EcoRI fragment for the presence of the major rRNA promoter(s) without success (Feingold et al., 1985). The upstream 4.2 kb EcoRI fragment was isolated from Cc5507 and subcloned into the low copy number plasmid pRK290 (Ditta et al., 1980). In order to isolate a restriction fragment that overlapped both the region 5’ to the 16 S rRNA gene and the 16 S rRNA gene itself, a 5.1 kb BglII-BglII restriction fragment was isolated from Cc5507 and subcloned into pRK290.

Figure

l(a)

shows

a restriction

map of the region covering approximately the 16 S rRNA gene.

enzyme

4 kb 5’ to

t Abbreviations used: kb, lo3 base-pairs; ORF, open reading frame, IR, inverted repeat; DR, direct repeat;

bp, base-pair(s).

Conserved Elements Upstream and Downstream (b) DNA sequencing strategy and nucleotide sequence Figure l(b) shows the strategy used to determine the nucleotide sequence of approximately 1300 nucleotides 5’ to the 16 S rRNA gene. Two smaller restriction fragments from the region 5’ to the 16 S rRNA gene were first isolated from pRK290 derivatives that contained larger restriction fragments covering this region (mentioned above). The smaller restriction fragments were the 08 kb SalI-EcoRI and the 67 kb BamHI-BamHI restriction fragments that overlapped each other by 120 nucleotides. They were subcloned into M13mp18 and M13mp19 for DNA sequence analysis. The 07 kb BamHI-BamHI fragment contained approximately 70 nucleotides of the 5’ end of the 16 S rRNA gene. The deletion subcloning method of Dale et al. (1985) was used to generate smaller subclones from the initial M13mp18 and M13mp19 clones in order to obtain overlapping nucleotide sequences. Figure 2 shows the nucleotide sequence of approximately 1280 nucleotides 5’ to the region coding for the mature 16 S rRNA gene. (c) Analysis of the nucEeotide sequence 5’ to the 16 S T-RNA gene The start of the 16 S rRNA gene was assigned (Feingold et al., 1985) to nucleotide 1279, on the basis of sequence homology between the C. crescentus 16 S rRNA gene (Feingold et al., 1985) and the E. coli rrnB 16 S rRNA (Brosius et al., 1981). Approximately 25 nucleotides in front of the 5’ end of the 16 S rRNA gene is one part of an inverted repeat sequence (Feingold et al., 1985) that could form a stem structure with the other part of the inverted repeat sequence located 3’ to the 16 S rRNA gene. This stem structure is the putative substrate for cleavage and processing of the 16 S rRNA by C. crescentus RNase III (Bellofatto et al., 1983). We have reported the nucleotide sequence up to the EcoRI restriction site located 399 nucleotides 5’ to the 16 S rRNA gene (Feingold et al., 1985). Although the previous nucleotide sequence was determined from C. crescentus strain CB13, there was 90% or greater homology with the nucleotide sequence from C. crescentus strain CB15 between the proximal EcoRI site and the BamHI site located within the 5’ end of the 16 S rRNA gene. The slight difference in the nucleotide sequence in this region may be the result of species differences. The location of some restriction sites in this region, for example EeoRI and DdeI, was identical in the two species. Examination of the nucleotide sequence in the 5’ flanking region of the 16 S rRNA gene revealed the presence of four small open reading frames (ORFs). One ORF began at nucleotide 101 and ended at nucleotide 419. Several nucleotides in front of the beginning of this ORF is a possible ribosomebinding site (Shine & Dalgarno, 1975). Another ORF began at nucleotide 195 and ended at 534. There does appear to be a possible ribosome-binding site immediately in front of this ORF. Two smaller

(a)

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Figure 1. Restriction map of the region 5’ to the C. crescentusrRNA gene cluster. (a) Diagram at the top of the Figure shows the location and arrangement of the 16 S rRNA gene, tRNA spacer region, and a portion of the 23 S rRNA gene in the region of Cc5507 examined (Ohta & Newton, 1981). Below is shown the restriction map in this region of Cc5507 and the location of some of the restriction enzyme sites 5’ to the 16 S rRNA gene. (b) Nucleotide sequencing strategy of the region 5’ to the 16 S rRNA gene. A portion of the 16 S rRNA gene that was sequenced is shown on the right of the restriction map. The arrows above and below the restriction map indicate the direction and extent of sequencing in these regions. (c) DNA restriction fragments used as probes to map the transcription start sites by S, nuelease protection assay. The asterisks show the position of the label on the fragment. B, BumHI; Bg, BgZII; D, DdeI; R, EcoRI; S, SalI.

ORFs were found downstream from the two previously mentioned. One began at nucleotide 424 and ended at nucleotide 643 and one other started at nucleotide 642 and ended at nucleotide 822. Neither of these two later ORFs appear to have an obvious ribosome-binding site. Most of the ORFs were found in different reading frames and were located more than 450 nucleotides upstream from the 16 S rRNA gene. A search of GenBank did not reveal any significant homologies with the four ORFs upstream from the 16 S rRNA gene. Further examination of the nucleotide sequence upstream from the 16 S rRNA gene revealed a number of inverted repeat (IR) and direct repeat (DR) nucleotide sequences. One inverted repeat sequence (IRl) immediately followed the ORF that ended at nucleotide 419 (ORFl). The possible significance of this IR sequence will be discussed in a later section. There were three sets of DR sequences found in the 5’ flanking region. Two sets of the DR sequences, DR2 and DR3, contained predominantely cytosine and guanosine bases and were somewhat homologous with each other.

K. Amemiya

248 10

Sal I *1

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------------------

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rRNA

TCBT~ACTAf~~TACCTGGnCT66mmCCCCf66~~CT~C(YITGT~AC~~rrsATCCTGG Barn HI CTMWC6AACXT66C66W6CCTMCACAT6WWC6tWC66ATCC

Figure 2. Nucleotide sequence of the region 5’ to the C. crescentus 16 S rRNA gene. The location of pertinent restriction enzyme sites-is shown above the sequence. Inverted repeat (IR) and direct repeat (DR) nucleotide sequences are indicated by horizontal arrows. The boxed nucleotides mark the beginning and end of an ORF. The wavy underline indicates a possible ribosome-binding site. The vertical arrows indicate the transcription initiation sites with the height of the arrow representing the relative frequency of initiation. The sequencesanalogous to the E. coli promoter consensus sequence (Hawley & McClure, 1983) are labeled - 10 and -30. The broken line above the nucleotide sequence in front of the 16 S rRNA-gene is part of an inverted repeat sequence surrounding the 16 S rRNA gene (Feingold et al., 1985).

(d) Determination of transcription start sites by S 1 nuclease analysis We had already shown that there were no transcription start sites or RNA polymerase-binding sites in the region between the 16 S rRNA gene and the proximal EcoRI restriction site 300 bp 5’ to the 16 S rRNA gene (Feingold et al., 1985). We now examined the region 5’ to the proximal EcoRI restriction site for the presence of transcription initiation sites by S, nuclease protection assays. Three restriction fragments were used as hybridization probes,in the S1 nuclease assays that covered over 1 kb 5’ to the 16 S rRNA gene. When the distal 06 kb SaZI-EcoRI restriction fragment (see Fig. 1 and 2) labeled at the 5’ end of the EcoRI site was used as the hybridization probe, a 260 base S1 nuclease-resistant product was obtained (Fig. 3(b)). A smaller 665 kb SalI-BamHI probe 5’-labeled at the BamHI site protected two fragments (Fig. 3(a), lane 1): (1) a large .heterogeneous product located 105 bases from the BamHI site; and (2) a smaller less heterogeneous S1 protected product located approximately 90 bases from the BamHI site. Finally, when a 636 kb BamHI-DdeI fragment labeled at the 5’ end of the DdeI site was used as the probe was hybrization probe, the complete

protected from S1 nuclease digestion (Fig. 3(a), lane 3). This later probe contained the 917 kb EcoRI-EcoRI restriction fragment just 5’ to the 16 S rRNA gene. These results suggest that there is only one major, and possibly one minor, transcription

start

site for this rRNA

gene cluster

located

approximately 735 nucleotides upstream from the 16 S rRNA gene. In order to determine the transcription initiation sites more precisely, S, nuclease protected products were examined on a DNA sequencing gel. The probe used in this experiment was a 665 kb SalI-BamHI fragment labeled at the 5’ end of the BamHI site. To determine if different growth conditions had an effect on the transcription of the rRNA gene cluster, total

RNA

was

prepared

from

cells

grown

in

complex medium (PYE), where the cell doubling time is approximately 90 to 100 minutes, and from cells grown in a defined minimal medium (M2), where the cell doubling time is approximately 150 to 180 minutes. The results show that there were at least two differences in the transcription of the rRNA gene clusters under these two different growth conditions (Fig. 4). When RNA obtained from cells grown in M2 medium was used in the S1 nuclease assay, a number of protected fragments (Pl) was obtained as well as several protected

Conserved Elements Upstream and Downstream

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(bl (0)

(b)

Figure 3. S1 n&ease

mapping of the transcription start sites of the rrnA transcription unit. Total RNA was extracted from C. crescentus CB15 grown in minimal M2 medium and used in S, nuclease protection assays as decribed in Materials and Methods. Double-stranded DNA fragments were labeled at the 5’ end of the sense strand and hybridized to 100 pg of total RNA and treated with S, nuclease. (a) Lane 1 shows S, nuclease-resistant products with the 0.65 kb SalI-BamHI DNA probe labeled at the BumHI site (see Figs 1 and 2). Lane 2 shows the results with the 0.65 kb &WI-BumHI probe without RNA. Lane 3 shows S, nuclease-resistant products with the 0.36 kb BumHI-DdeI DNA probe labeled at the DdeI site. Lane 4 shows the results with the @36 kb BarnHI-DdeI probe without RNA. (b) Lane 1 shows S, nuclease-resistant products with the @8 kb SalI-EcoRI DNA probe labeled at the EcoRI site. S, nuclease-resistant products are labeled with asterisks. Lanes marked M denote DNA size markers.

(P2) approximately ten nucleotides downstream (Fig. 4(b)). In contrast, when RNA from cells grown in rich medium wzw used in the S,

products

Figure 4. S, n&ease

mapping of the transcription start sites of the rrnA transcription unit. Total RNA was extracted from C. crescentus CB15 grown in PYE or minimal M2 medium and used in S1 nuclease protection assays as described in Materials and Methods. The @65 kb SalI-BarnHI DNA fragment labeled at the 5’ end of the BarnHI site was used with 100 pg of total RNA. (a) Lane 1 shows S, nuclease-resistant products when RNA from cells grown in PYE was used. (b) Lane 1 shows products when RNA from cells S1 nuclease-resistant grown in minimal M2 medium was used. Lanes 2 and 3 in (a) and (b) show Maxam & Gilbert (1980) reactions G and G+A, respectively, for size markers. Nucleotides of the sense strand are labeled on the right of (b). Pl and P2 refer to the major and minor start sites, respectively, with the predominant S, products labeled with an arrow.

nuclease assay, only the large heterogeneous protected products (Pl) were obtained. In addition, there appeared to be a relative change in the use of the first initiating nucleotide. These differences e&n be seen more clearly in the scans of the autoradiogram of the S1 nuclease-protected products (Fig. 5). There was an approximately 50% decrease in the

of nucleotide

promoter)7

TT

AGAAG

GGGTT

GTGGG

GCCAG

-30

CTACA

TTGGC

TCGCC

sequences near the transcription

CACGT

CCNP5

GGCTG

GCCGA

GCTAC

p-TTG

G YCGRC

-20 .

AATGG

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YARAT

CTAGA

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sequencet

CC

cGACT

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TC

BCAGA

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GCCTC

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T

site of C. crescentus transcription

Nucleotide

initiation

Table 1

GGGCT

CGGAA

GCTCG

CGAAC

CG

GAACG

+10

units/promoters

ccc:

t Nucleotide sequences have been aligned relative to the transcription start site. Dot above a nucleotide marks the initiating nucleotide(s). Underlined nucleotides mark sequences that are analogous to sequences found in E. coli promoters at positions - 10 or - 35 relative to the transcription start site (Hawley & McClure, 1983). $ From this paper. The boxed region shows homologous nucleotides. The arrows above the nucleotides indicate DR sequences. The wavy underline indicates nucleotides implicated in the stringent response of rRNA expression in E. coli (Travers, 1984). Q From Ross & Winkler (198%). 11From Mullin et al. (1987). 7 From Amemiya et al. (1986).

16 S rRNA (internal

wmemus:

unit/promoter

jh (consensus)ll

trp FRA§

rrnA Pl$

Transcription

Comparison

251

Conserved Elements Upstream and Downstream first major initiating nucleotide of Pl in cells grown in M2 medium when compared to Pl of cells grown in complex medium. The minor set of S,-protected products (P2) is approximately 12% of the predominant (Pl) S1-protected products. There also appears to be an additional minor initiating nucleotide when RNA from cells grown in PYE was used. These results indicate that the transcription start site for the rRNA gene cluster is heterogeneous and, depending on the growth conditions, can initiate from one and possibly another minor start site. Furthermore, the relative amount of initiation from the first nucleotide at the major start site is altered under different growth conditions. Comparison of the nucleotide sequence from several C. crescentus promoters whose transcription start sites have been determined has allowed them to be grouped in several categories based on the presence of conserved nucleotide sequences (see Table 1). One category includes the $a gene promoters, which have been shown to be under regulation. A conserved nueleotide periodic sequence has been found in the promoter region of these genes at positions (relative to the transeription start site) - 13, -24 and -100 (Chen et al., 1986; Mullin et aE., 1987). The promoter located within the 16 S rRNA gene would be in another category (Feingold et al., 1985; Amemiya et al., 1986). This promoter is recognized in vitro by both C. crescentus and E. coli RNA polymerase and has been shown to be recognized in vivo in E. coli. Not surprisingly, this promoter has the conserved nucleotide sequences at positions - 10 and -35 similar to that found in E. coli promoters read by the E. coli RNA polymerase containing cr7’ (Hawley & McClure, 1983). Another category would contain the promoters for the QFBA operon (Ross & Winkler, 1988a) and the rrnA PI promoter. Promoters in this group appear to respond to changes in growth conditions, such as growth in rich versus minimal medium (Ross & Winkler, 1988b). In addition, there appears to be a striking homology in several additional regions in the rrnA Pl and trpFBA promoters (Table 1). Centered approximately around the - 10 region of both promoters is a 12 bp nucleotide sequence 5’-GYCGRCYARATC-3’ not present in the other reported Caulobacter promoters. Also, further upstream from both promoters are two to three similar 7 bp DR units, 5’-TCGCCRR-3’, with one of the DR units being part of the 12 bp homologous nucleotide sequence found centered around the - 10 region. Both the rrnA PI and tr;pFBA promoters also have a nucleotide sequence similar to that found in the -35 region of the E. coli RNA polymerase 07’ promoters, except that it is located near t,he - 30 region. Surprisingly, another identical nucleotide sequence found in both the rrnA Pl and trpFBA promoters is an octamer (5’-TCGGAACG-3) located just downstream from the transcription start site. This octamer sequence does not appear to be present in the$a genes. It is difficult to define the &s-acting elements that

G

C G

AT

4-t

(b) Figure 5. Scans of autoradiograms of S, nuclease-resistant products from Fig. 4. (a) Scan of S, nuclease-resistant products from S, nuclease assay using RNA from cells grown in PYE medium. (b) Scan of S1 nuclease-resistant products from S, nuclease assay using RNA from cells grown in minimal M2 medium. Peaks are labeled with the corresponding nucleotide of the sense strand.

could serve as the promoter for P2, because of its proximity to rrnA Pl. Many of the putative cisacting sequences found near the start site for the P1 promoter would overlap the promoter sequence for P2. Overlapping the start site of Pl, however, is the nucleotide sequence 5’-CCGCC-3’, which is not

252

K. Amemiqa

T

G

T

G c.

G

G@ c c.

C

G

l

G

G.

c

A.

T

G.

C

nG=-276

kcal

5’- C C G . C A C -3’ Figure upstream start site. structure by Tinoco

6. Potential stem-loop structure located from the start site of the rrnA PI transcription The free energy (66) of the potential stem-loop was determined according to the rules proposed et al. (1973).

1 P2

found at the start sites of P2 or the trpFBA promoter. This pentamer sequence is similar to the consensus sequence found near the start site of some of the promoters of the rRNA operons in E. co&. It has been proposed by Travers (1984; Lamond, 1985) to be a “discriminator” sequence element that may be responsible for the stringent response exhibited in E. coli. The stringent response is characterized by the down regulation of the synthesis of stable RNA in response to amino acid deprivation (Nomura et al., 1984).

Between the transcription start sites and the 16 S rRNA gene, several IR sequences and DR sequences were found. Whether these putative stem and loop structures and direct repeat elements affect the expression and/or processing of rRNA transcripts remains to be further examined. One IR sequence was found approximately 100 bases upstream from the major transcription start site (Figs 2 and 6). It could form a 9 bp stem structure with a four base loop and would have a AG of -27.6 kcd (1 cal = 4.184 J: Tinoco et aE., 1973) and could possibly act as a rho-dependent termination site, since it was not followed by an oligo(T) sequence characteristic of rho-independent termination sites (Platt, 1986).

1234567 Figure 7. S, nuclease mapping

of the transcription start sites of the rrnA transcription unit during the C. crescentus cell cycle. Total RNA was extracted from synchronously growing cells at specified times and used in S,nuclease protection assays as described in Materials and Methods. The 0.65 kb SalI-BumHI DNA fragment labeled at the 5’-end of the BumHI site was used as the probe with 100 pg of total RNA. RNA was extracted from cells at the following periods: lane 1, 0 min; lane 2, 60 min; lane 3, 120 min; lane 4, 150 min; and lane 5, 180 min. Lanes 6 and 7 are Maxam & Gilbert (1980) reactions G and G + A, respectively. The figure above each lane reflects the developmental stage of the cell at the specified periods. Brackets to the right of lane 7 denote initiating nucleotides of Pl and P2.

(e) Expression of the rRNA gene cluster during the cell cycle

S1 nuclease protection assays were used to determine if expression from the major and the possible

Conserved Elements Upstream and Downstream minor start sites of the rRNA gene cluster was altered during the C. crescentus cell cycle. Total RNA was extracted from synchronously growing cells in M2 minimal medium at different periods of the cell cycle. Equal amounts of RNA from these different periods were hybridized to the 0.65 kb SalI-BamHI restriction fragment labeled at the 5’ end of the BamHI site before S, nuclease digestion. Figure 7 shows the results of the S1 nuclease protection assay with the cell cycle RNA. Heterogeneous S1 nuclease-resistant products from the major start region were seen at all stages of the cell cycle. Very few S, nuclease-resistant products were obtained from the minor start site with RNA isolated from swarmer cells. We only begin to see S1 nucleaseprotected fragments from the minor start region with RNA from stalk cells after 60 minutes of incubation and with RNA obtained from cells at later stages of the cell cycle. These results suggest that transcription from the major start site is continuous throughout the cell cycle, but that possibly transcription from the minor start site does not occur in the swarmer cell.

4. Discussion The transcription start sites for one of the two rRNA gene clusters of C. crescentus have been located more than 700 nucleotides upstream from the 16 S rRNA gene. For the sake of clarity, this rRNA gene cluster will be referred to as the rrnA gene cluster hereinafter. There is a major start site (Pl ) from which expression occurs in either complex or minimal defined media, and possibly a minor transcription start site (P2) that is seen only in minimal medium. Transcription from the major start site did not appear to be regulated in a temporal manner during the cell cycle. Expression from the minor start site, however, was detected during the developmental cell cycle at all stages except in the swarmer cell. There are a number of possible explanations for observing the appearance of the minor start site in cells grown only in minimal medium. One reason may be that expression from P2 may be repressed in cells grown in complex media. On the other hand, it may be that expression from Pl is increased to a level where expression from P2 is difficult to detect in cells grown in rich media. This later case is reminiscent of the rRNA expression in E. coli where Pl responds to changes in the growth medium, while P2 expression remains relatively unaffected regardless of the type of growth media (Nomura et al., 1984). Also in this regard, it is possible that only one type of cell is responding to changes in the growth medium, as reflected by a change in rRNA expression. We know that a specific function, such as DNA replication, does occur only at specific cell type (stalk). Finally, there is a possibility that transcripts that come from the minor start site could actually be the result of cleavage or processing of transcripts from the major start site. Further studies must be carried out to

253

determine which of these possibilities can account for the differences that we see in transcription of the rrnA gene cluster in different media and in different cell types. The finding of a consensus sequence in the promoters of the jia genes (Mullin et al., 1987) and now for a different set of conserved sequences elements in the promoters of the rrnA and trpFBA genes in Caulobaeter suggests that there may be more than one form of RNA polymerase used to transcribe this diverse set of genes. This possibility is strengthened by two other findings. First, a set of heat-inducible proteins could be observed upon heat shock in Caulobacter (Gomes et al., 1986; Reuter & Shapiro, 1987). It has been demonstrated in E. coli that the heat shock response is characterized by an increase in the level of an alternative cr subunit that increases the rate of transcription of a specific set of proteins (Grossman et al., 1984). The promoters of the transcription units that code for these proteins share a common consensus sequence that is recognized by the heat-inducible o subunit (Cowing et al., 1985). Secondly, the nucleotide sequence and transcription start site of the C. crecentus$aE gene has been determined (Kaplan et al., 1989), and sequence elements within the promoter region appear to be similar to the Bacillus subtilis g** promoter eonsensus sequence (Gilman et al., 1981). It has been shown that the B. subtilis az8 is required for flagellar synthesis (Helmann & Chamberlin, 1987). Examination of Jla and the genes of E. co& and Salmonella typhimurium has revealed the presence of sequence elements upstream from the transcri tion start site that match that of the B. subtilis a !L promoter (Helmann & Chamberlin, 1987). Because of the presence of the common sequence elements in these genes, it has been proposed that an a alternative factor regulates the expression of theJa and the genes in E. coli and S. typhimurim as well as those in B. subtilis. In B. subtilis the existence of four other a factors required for the growth of vegetative cells and development of endospores has been well documented (Losick & Pero, 1981; Losick et al., 1986). It has been demonstrated that the temporal regulation of flagellin and hook genes in C. crescentus is at the level of transcription (Sheffery & Newton, 1981; Milhausen & Agabian, 1983; Champer et al., 1985; Ohta et al., 1985; Chen et al., 1986; Minnich &, Newton, 1987). Also, it has been shown that the expression of specific $a genes in C. crescentus is under positive regulation by other $a t,ranscription units (Mullin et al., 1987). Whether one or more of the positive regulators affect transcription by DNA-protein interaction or by modification of the transcriptional specificity of RNA polymerase remains to be elucidated. However, the use of transcription units with promoters containing different &s-acting sequence elements, allows the identification in vitro of alternate a-like factors. The ability to separate morphologically distinct cell types where positive regulation by trans.acting factors may be responsible for differential gene expression

K. Amemiva

254

makes C. crescentus a model approach biochemically.

system

to pursue

this

The author thanks Dr Lucy Shapiro for her help and support while this work was being carried out in her laboratory. In addition, the author thanks both Dr Lucy Shapiro and Ruth Bryan for their help in the preparation of this manuscript. The author also thanks Susan Roberts, Zi Cun Lai, Jeff Kaplan, and especially Peter Ruvolo, for introducing him to DNA sequencing. Thanks is also given to Sandy Reuter for her assistance in the cell synchrony experiments. Finally the author would like to thank Jim Garbern for his assistance in the computer search and Nanette Whitaker for her excellent typing. A portion of this work was performed in the Department of Molecular Biology, Albert Einstein College of Medicine, Bronx, NY.

References Amemiya, K., Raboy, B. & Shapiro, L. (1980). Virology, 104, 109-l 16. Amemiya, K., Bellofatto, V., Shapiro, L. $ Feingold, J. (1986). J. Mol. BioZ. 187, 1-14. Bellofatto, V., Amemiya, K. & Shapiro, L. (1983). J. Biol. Chem. 258, 5467-5476. Berk, A. J. & Sharp, P. A. (1977). Cell, 12, 721-732. Brosius, J., Dull, T. J., Sleeter, D. & Noller, H. F. (1981). J. Mol. Biol. 148, 107-127. Champer, R., Bryan, R., Gomes, S. L., Purucker, M. & Shapiro, L. (1985). Cold Spring Harbor Symp. Quant. Biol. 50, 831-840. Chen, L.-S., Mullin, D. & Newton, A. (1986). Proc. Nat. Acad. Sci., U.S.A. 83, 2860-2864. Contreras, I., Shapiro, L. & Henry, S. (1987). J. Bacterial. 135, 1130-l 136. Cowing, D. W., Bardwell, J. C. A., Craig, E. A., Woolford, C., Hendrix, R. W. & Gross, C. A. (1985). Proc. Nat. Acad. Sci., U.S.A. 82, 2679-2683. Dale, R. M. K., McClure, B. A. & Houchins, J. P. (1985). Plasmid, 13. 31-40. Ditta, G., Stanfield, S., Corin, 1). & Helinski. D. R. (1980). Proc. Nat. Acad. Sci., U.S.A. 77, 734777351. Evinger, M. & Agabian, N. (1977). J. Bacterial. 132, 294-301. Feingold, J., Bellofatto, V., Shapiro, L. & Amemiya, K. (1985). J. Bacterial. 163, 155-166. Fujita, N., Nomura, T. & Ishihama, A. (1987). J. Biol. Chem. 262, 1855-1859. Gill, P. & Agabian, N. (1983). J. Biol. Chem. 258, 739557401. Gilman, M., Wiggs, J. & Chamberlin, M. J. (1981). NucZ. Acids Res. 9, 5991-6000. Gomes, S. L., Juliani, M. H., Maia, J. C. C. & Silva, A. M. (1986). J. Bacterial. 168, 923-930. Grossman, A. D., Erickson, J. W. & Gross, C. A. (1984). Cell, 38, 383-390. Hahnenberger, K. & Shapiro, L. (1987). J. Mol. Biol. 194, 91-103. Hawley, D. & McClure, W. (1983). Nucl. Acids Res. 11, 223772255. Edited

Helmann, J. D. & Chamberlin, M. J. (1987). Proc. Nat. Acad. Sci., U.S.A. 84, 6422-6424. Hirschman, J., Wong, P.-K., Sei, K., Keener, J. & Kustu, S. (1985). Proc. Nat. Acad. Sci., U.S.A. 82, 7525-7529. Hunt, T. P. & Magasanik, B. (1985). Proc. Nat. Acad. Sci., U.S.A. 82, 8453-8457. Kaplan, J. B., Dingwall, A., Bryan, R., Champer, R. & Shapiro, L. (1989). J. Mol. Biol. 205, 71-84. Lamond, A. I. (1985). Trends B&hem. Sci. 10, 271-274. Losick, R. & Pero, J. (1981). Cell, 25, 582-584. Losick, R., Youngman, P. & Piggot, P. J. (1986). Annu. Rev. Genet. 20, 625-669. Maniatis, T., Fritsch, E. & Sambrook, J. (1982). Editors of Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Maxam, A. & Gilbert, W. (1980). In Methods in EnzymoEogy (Wu, R., ed.), vol. 65, pp. 4499520, Academic Press, New York and London. Milhausen, M. & Agabian, N. (1983). Nature (London), 302, 63&632. Milhausen, M., Gill, P., Parker, G. & Agabian, N. (1982). Proc. Nat. Acad. Sci., U.S.A. 79, 6847-6851. Minnich, S. A. & Newton, A. (1987). Proc. Nat. Acad. Sci., U.S.A. 84, 1142-1146. Mullin, D., Minnich, S. A., Chen, L.-S. & Newton, A. (1987). J. Mol. Biol. 195, 939-943. Newton, A., Ohta, N., Huguenel, E. & Chen, L.-S. (1985). In Molecular Biology of Microbial Differentiation (Letlow, P. & Hoch, J., eds), pp. 267-276, Amer. Sot. Microbial., Washington, DC. Nomura, M., Gourse, R. & Baughman, G. (1984). Annu. Rev. Biochem. 53, 75-117. Ohta, N. & Newton, A. (1981). J. Mol. Biol. 153,291-303. Ohta, N. & Newton, A. (1984). J. Bacterial. 158, 897-904. Ohta, N., Chen, L.-S. & Newton, A. (1982). Proc. Nat. Acad. Sci., U.S. A. 79, 4863-4867. Ohta. N., Chen, L.-S., Swanson, E. 6 Newton, A. (1985). J. Mol. Biol. 186, 107-115. Pharmacia (1984). Manual for M 13 cloning/sequencing system. Platt, T. (1986). Annu. Rev. Bioehem. 55, 339-372. Poindexter, J. S. (1964). Boxteriol. Rev. 28, 231-295. Purucker, M.. Bryan, R., Amemiya, K., Ely, B. & Shapiro, L. (1982). Proc. Nat. Acad. Sci., U.S.A. 79, 679776801. Reuter S. $ Shapiro. L. (1987). J. Mol. Biol. 194: 653-662. Ross, C. t Winkler, M. (1988a). J. Bacterial. 170, 757-768. Ross, C. & Winkler, M. (1988b). J. Bacterial. 170, 769-774. Sanger, F., Nicklen, S. & Coulson, A. R. (1977). Proc. Nat. Acad. hi., U.S.A. 74, 5463-5467. Shapiro, L. (1985). Annu. Rev. Cell Biol. 1, 173-207. Sheffery, M. & Newton, A. (1981). Cell, 24, 49-57. Shine, J. & Dalgarno, L. (1975). Nature (London), 254, 34-38. Sollner-Webb. B. & Reeder, R. H. (1979). Cell, 18. 485-499. Tinoco, I., Borer, P. N., Dengler, B., Levine, M. D., Uhlenbeck, 0. C., Crothers, D. M. & Gralla, J. (1973). Nature New Biol. 246, 40-41. Travers, A. A. (1984). Nucl. Acid. Res. 12, 2605-2618.

by R. Schlief