Promoter activity and transcript mapping in the regulatory region for genes encoding ribosomal protein S15 and polynucleotide phosphorylase of Escherichia coli

Abstract

The genes encoding ribosomal protein S15 (rpsO) and polynucleotide phosphorylase (pnp) occupy adjacent positions and are oriented in the same direction on the Escherichia coli chromosomes. The nucleotide sequence of the region controlling the expression of these two genes has been determined. Two in-phase gene fusions between pnp and lacZ were constructed. The fusions define the translational reading frame of the pnp gene and indicate that the expression of pnp is independent of the upstream rpsO gene. Transcript mapping with nuclease S1 demonstrated that the two genes are transcribed from separate promoters and that the rpsO-pnp intergenic space contains a strong transcriptional terminator. The transcriptional start points have been localized.

Fig. 1

Structures of plasmids pMC1403, pHE1 and their derivatives. The plasmid pMC1403 contains a 6.8-kb EcoRI-SalI fragment carrying a 5′-deleted lacZ gene (‘lacZ) inserted between the EcoRI and the SaiI sites of pBR322. The plasmid pHE1 carries a 4.8-kb HindIII-EcoRI fragment from near 68 min on the E. coli chromosome that encodes ribosomal protein S15 (rpsO) and PNPase (pnp) (see solid bars). Open bars represent lacZ coding sequences. The positions of various Tn5 insertions used to position and orient the pnp gene are indicated. Restriction site abbreviations are: E, EcoRI; S, SmaI; B, BamHI; H, HpaI; T, TaqI; P, PstI and A, AccI; the centered dots indicate polylinker sequences; the slash symbols indicate ligation of specified sites. The dashed lines indicate pBR322 vector sequences. Constructions of the plasmids pUB2, pMS31, pSH122 and pMB1 were as follows. Plasmid pMS31 is a rpsO-lacZ fusion and was constructed by inserting the 262-bp HpaI[3]-PstI[1] fragment from pHE1 into the SmaI site of pMC1403. A short connector PstI-SmaI fragment obtained from pUC9 was used to fuse the PstI site on the fragment to the SmaI site in front of lacZ. This short connector fragment results in an out-of-frame fusion between the PstI site of rpsO and the SmaI site of lacZ (Fig. 3). Plasmid pSH122 is a pnp-lacZ fusion and was constructed by inserting the 843-bp HpaI[3–4] fragment into the SmaI site of pMC1403. One of these, pSH122, was shown by DNA sequence analysis to be an in-frame pnp-lacZ fusion (Fig. 3). This plasmid thus contains an active copy of the intact rpsO gene and an in-frame pnp-lacZ gene fusion. The plasmid pMB1 was constructed by inserting the 588-bp PstI[l]-BamHI fragment from pSH 122 between the SmaI and the BamHI site of pMC1403. The junction between the vector SmaI site and the fragment PstI site contains a short connector fragment from pUCl 3. The connector fragment contains a blunt end HindIII site (filled in with PolIk) at one end ligated to the vector SmaI site and a PstI site at the other end ligated to the PstI of the fragment. This plasmid contains only the distal half of rpsO and the in-frame pnp-lacZ fusion. A fourth plasmid, pUB2, is a derivative of pUC8 and was used as a source of DNA for some of the S1 nuclease transcript mapping experiments. The 900-bp TaqI-PstI fragment from pHE1 was inserted between the AccI and PstI sites in pUC8.

Fig. 2

Nucleotide sequence of the 5′ controlling regions of the rpsO and pup genes. The nucleotide sequence of the HpaI[3–4] fragment from pHEI was determined (Sanger, 1976; Maxam and Gilbert, 1980). The coding sequence of rpsO extends from nt 148 to nt 417. The coding sequence of pup has not been unambiguously established but a likely candidate is the ORF with a TTG translation start at nt 663. The PstI at nt 262 and the HpaI site at nt 843 were used to construct rpsO and pnp fusions with lacZ. Some of the restriction sites used for S1 nuclease mapping of the rpsO and pnp gene transcripts are HpaII (212), PstI (262) and HpaII (721). The major transcription start and stop points for the in vivo S15 mRNA occur at about nt 46 and 458 (arrowhead and dot), respectively. The major start point for the in vivo PNPase mRNA occurs at about nt 583 (arrowhead). Sequences related to the consensus −10 and −35 sequences for RNA polymerase promoter function immediately upstream from the major start points are indicated. This entire sequence is identical to that of Portier and Regnier (1984) except that their sequence contains a single insertion of a C residue between nt 617 and 618.

Fig. 3

Nucleotide sequences of the rpsO and pnp fusion junctions with lacZ. The construction of plasmid pMS31, pSH122 and pMB1 is described in Fig. 1. The pMS31 fusion is out-of-phase whereas pSH122 and pMB1 are in-phase.

Fig. 4

The transcription patterns of the rpsO and pnp genes. The results of the S1s nuclease transcript mapping experiments illustrated in Figs. 5,6 and 7 are summarized. At the top the DNA structure is depicted and relevant nt positions are identified. Restriction sites are: HpaII, 212 and 721; PstI, 262; BamHI, 848 (in lacZ). The major promoters (P) and terminators (T) are indicated. The end-labeled DNA probes (open boxes) are: A, 5′ end-labeled 900-bp BamHI-PstI fragment derived from plasmid pUB2; B, 3′ end-labeled 509-bp HpaII fragment from plasmid pHE1; C, 5′ end-labeled 509-bp HpaII fragment, and D, 5′ end-labeled 590-bp PstI-BamHI fragment from plasmid pSH122. Shaded boxes: major protected fragments (sizes in nt); dashed lines: minor protected fragments. Downward arrowheads over the read-through transcripts between rpsO and pnp: possible RNA-processing site.

Fig. 5

Nuclease S1 mapping of the 5′ ends of the S15 gene transcript. The 900-bp BumHi-PstI fragment isolated from plasmid pUB2 and containing about 650 bp of 5′-flanking sequence in front of the S15 gene was 5′ end-labeled with polynucleotide kinase and [γ-³²P]ATP. The Pstl end of the fragment is at nt 263 within the coding region of the S15 gene and the Bam HI is from within the vector DNA sequences and is therefore never protected. The labeled fragment was hybridized to 5 μg RNA. digested with S1 nuclease, denatured and electrophoresed on an 8°, PA sequencing gel. The RNAs used for each sample are: (A) E. coli ribosomal RNA; (B)MC1000 RNA; (C)MC1000[pMBI] RNA;(D)MC1000[pSH122] RNA; (E) MC1000[pHEI] RNA; (F) MC1000[pMS31] RNA; last lane, M_T standards. The sizes in nt of major and minor S1 protected fragments are indicated. The M _T standards were generated by 3′ end-labeling HpaII cut pBR322 with PoIIk and [α-³²P]dCTP. The standard lengths in nt from top to bottom are 622, 527, 404, 309, 242, 238, 217, 201, 190, 180, 160, 147, 122, 110, 90, 76, 67, 34, 26, 15 and 9. The position of the largest fragment is marked by an arrowhead. The left portion of the figure is a short and the right portion a long exposure of the autoradiogram.

Fig. 6

Nuclease S1 mapping of the 3′ end of the S15 gene transcripts. A 509-bp HpaII fragment (nt 212–721) isolated from plasmid pHE1 and containing the distal portion and the 3′-flanking region of the S15 gene was 3′ end-labeled with PolIk and [α-¹²P]dCTP. The fragment was hybridized to 5 or 10 μg of RNA, digested with S1 nuclease, denatured and electrophoresed on an 8°, PA sequencing gel. The samples are: (A) E. coli ribosomal RNA; (B and C) 5 and 10 μg of MC1000 RNA; (D) MC1000[pHE1] RNA: (E) MC1000[pMS31] RNA; (F) MC1000[pSH122] RNA; (G) MC1000[pMB1] RNA; (P) untreated end-labeled fragment. The sizes in nt of the major protected fragments are indicated. The M_T standards are as described in the legend to Fig. 5 and the 622-nt fragment is marked by an arrowhead.

Fig. 7

Nuclease S1 mapping of the 5′ ends of pnp gene transcripts. Two fragments, a 509-bp HpaII fragment (nt 212–721) isolated from plasmid pHE1 and a 590-bp PstI-BamHI fragment (nt 262–848) from plasmid pSH122, were 5′ end-labeled with polynucleotide kinase and [γ-³²P]ATP. The HpaII fragment extends from within the coding region of S15 to codon 21 in the pnp gene. The PstI-BamHI fragment extends from within the coding region of the S15 gene through the coding region of the pnp gene and ends at the BamHI site just within the lacZ coding sequence of the fusion gene. The labeled fragments were hybridized to RNA, digested with S1 nuclease, denatured and electrophoresed on an 8°, PA sequencing gel. The samples are: (A) 10 μg E.coli ribosomal RNA; (B and B′) 10 or 20 μg of MC1000 RNA; (C and C′) 5 or 10 μg of MC1000[pSH122] RNA; (D) 5 μg of MC1000[pMB1]. The right portion of the figure shows two exposures of the same autoradiogram. The 509-bp probe was used in the right and center panels and the 590-bp probe was used in the left panel.