SmpB, a unique RNA-binding protein essential for the peptide-tagging activity of SsrA (tmRNA).

In bacteria, SsrA RNA recognizes ribosomes stalled on defective messages and acts as a tRNA and mRNA to mediate the addition of a short peptide tag to the C-terminus of the partially synthesized nascent polypeptide chain. The SsrA-tagged protein is then degraded by C-terminal-specific proteases. SmpB, a unique RNA-binding protein that is conserved throughout the bacterial kingdom, is shown here to be an essential component of the SsrA quality-control system. Deletion of the smpB gene in Escherichia coli results in the same phenotypes observed in ssrA-defective cells, including a variety of phage development defects and the failure to tag proteins translated from defective mRNAs. Purified SmpB binds specifically and with high affinity to SsrA RNA and is required for stable association of SsrA with ribosomes in vivo. Formation of an SmpB-SsrA complex appears to be critical in mediating SsrA activity after aminoacylation with alanine but prior to the transpeptidation reaction that couples this alanine to the nascent chain. SsrA RNA is present at wild-type levels in the smpB mutant arguing against a model of SsrA action that involves direct competition for transcription factors.

Changing the mechanism of transcriptional activation by phage lambda repressor.

The first steps of transcription initiation include binding of RNA polymerase to a promoter to form an inactive, unstable, closed complex (described by an equilibrium constant, K(B)) and isomerization of the closed complex to an active, stable, open complex (described by a forward rate constant, k(f)). lambda cI protein activates the PRM promoter by specifically increasing k(f). A positive control mutant, cI-pc2, is defective for activation because it fails to raise k(f). An Arg to His change in the sigma70 subunit of RNA polymerase was previously obtained as an allele-specific suppressor of cI-pc2. To elucidate how the mutant polymerase restores the activation function of the mutant activator, abortive initiation assays were performed, using purified cI proteins and RNA polymerase holoenzymes. The change in sigma does not significantly alter K(B) or k(f) in the absence of cI protein. As expected, cI-pc2 activates the mutant polymerase in the same way that wild-type cI activates the wild-type polymerase, by increasing k(f). An unexpected and novel finding is that the wild-type activator stimulates the mutant polymerase, but not wild-type polymerase, by increasing K(B).

The insE open reading frame of IS1 is not required for formation of cointegrates.

The role of the insE open reading frame in transposition of IS1 was reexamined by using an insE nonsense mutation that does not alter the amino acid sequence of InsA inhibitor or InsAB transposase. The mutant was active in all strains tested, showing that insE is not essential for formation of cointegrates.

Probing the informational content of Escherichia coli sigma 70 region 2.3 by combinatorial cassette mutagenesis.

Combinatorial cassette mutagenesis was used to probe the informational content of region 2.3 of sigma 70, the RNA polymerase subunit that confers promoter specificity. Region 2.3 is highly conserved among major sigmas of diverse eubacteria, and has been predicted to have a role in melting the DNA duplex around the startpoint of transcription. This prediction was based on sequence similarity with the RNP-1 (ribonucleoprotein) motif of eukaryotic single-stranded RNA-binding proteins, and the abundance of aromatic and basic residues that could potentially interact with the single-stranded DNA. The mutagenesis technique used here consists of simultaneously mutagenizing several codons by cloning synthetic DNA cassettes, and characterizing the rare mutants that retain activity. The results show that most residues in region 2.3 are surprisingly tolerant of amino acid substitutions, including several conserved aromatics and other residues that match the RNP-1 motif. These conserved residues are not essential for transcription even at 17 degrees C, where the DNA melting step is more likely to be rate-limiting. In contrast, Thr429 is quite intolerant to substitution and is predicted to have an important role in sigma 70 function.

Target of the transcriptional activation function of phage lambda cI protein.

Activation of transcription initiation by the cI protein of phage lambda is thought to be mediated by a direct interaction between cl and RNA polymerase at the PRM promoter. Two negatively charged amino acid residues in the DNA binding domain of cI play a key role in activation, suggesting that these residues contact RNA polymerase. The subunit of RNA polymerase involved was identified by selecting polymerase mutants that restored the activation function of a mutant form of cI protein. Although previous studies suggest that several activators interact with the alpha subunit of RNA polymerase, the results here suggest that cI interacts with the sigma subunit. An arginine to histidine change near the carboxyl terminus of sigma specifically suppresses an aspartic acid to asparagine change in the activation region of cI. This finding supports the direct-contact model and suggests that a cluster of positively charged residues near the carboxyl terminus of sigma is the target of the negatively charged activation region of cI.

Use of electrophoretic mobility to determine the secondary structure of a small antisense RNA.

Natural antisense RNAs have stem-loop (hairpin) secondary structures that are important for their function. The sar antisense RNA of phage P22 is unusual: the 3' half of the molecule forms an extensive stem-loop, but potential structures for the 5' half are not predicted to be thermodynamically stable. We devised a novel method to determine the secondary structure of sar RNA by examining the electrophoretic mobility on non-denaturing gels of numerous sar mutants. The results show that the wild-type RNA forms a 5' stem-loop that enhances electrophoretic mobility. All mutations that disrupt the stem of this hairpin decrease mobility of the RNA. In contrast, mutations that change the sequence of the stem without disrupting it (e.g. change G.U to A.U) do not affect mobility. Nearly all mutations in single-stranded regions of the structure also have no effect on mobility. Confirmation of the proposed 5' stem-loop was obtained by constructing and analyzing compensatory double mutants. Combinations of mutations that restore a base-pair of the stem also restore mobility. The genetic phenotypes of sar mutants confirm that the proposed secondary structure is correct and is essential for optimal activity of the antisense RNA in vivo.

Hierarchies of base pair preferences in the P22 ant promoter.

Oligonucleotide-directed mutagenesis was used to complete a collection of mutations in the -35 and -10 hexamers of the ant promoter of Salmonella phage P22. The effects of all 36 single-base-pair substitutions on promoter strength in vivo were measured in strains carrying the mutant promoters fused to an ant-lacZ gene on a single-copy prophage. The results of these assays show that certain consensus base pairs are more important than others; in general, the least-critical positions are among the most poorly conserved. Some mutations within the hexamers have smaller effects on promoter strength than certain mutations outside the hexamers in this and other promoters. Several different patterns of base pair preferences are observed. These hierarchies of base pair preferences correlate well (but not perfectly) with the hierarchies defined by the frequency distribution of base pairs at each position among wild-type promoters. The hierarchies observed in the ant promoter also agree well with most of the available information on base pair preferences in other promoters.

Pseudo-templated transcription by Escherichia coli RNA polymerase at a mutant promoter.

A G----T mutation at the start-point of transcription of the phage P22 sar promoter (sar + 1T) causes a novel defect in promoter clearance by Escherichia coli RNA polymerase (RNAP) in vitro. Under standard transcription conditions, in the presence of high concentrations of all four NTPs, the predominant products from this promoter are poly(U) chains of varying length. Because the mutation creates a run of four T: A base-pairs from - 1 to +3 (TGTT----TTTT), we propose that synthesis of poly(U) is pseudo-templated by the A4 stretch on the template strand. G----A and G----C mutations at position +1 do not cause pseudo-templated transcription. Several molecules of poly(U) are produced and released per sar+1T promoter-polymerase complex without dissociation of RNAP from the template DNA. The exponential relationship between yield and size of individual poly(U) species indicates that there is a constant probability that another U residue will be added to the nascent chain. Presumably, pseudo-templated transcription occurs by a slippage (stuttering) mechanism like that proposed to explain certain kinds of RNA editing in eukaryotic viral mRNAs.

Changes in conserved region 2 of Escherichia coli sigma 70 affecting promoter recognition.

We describe a mutation in rpoD, the gene encoding the sigma 70 subunit of RNA polymerase, which alters the promoter specificity of the holoenzyme in vivo. The mutant sigma causes a substantial and specific increase in the activity of mutant ant and lac promoters with a T.A to C.G substitution at position -12, the first position of the -10 hexamer. The rpoD mutation is a single base-pair substitution causing a Gln----His change at position 437, which is in a domain of conserved region 2.4 that is predicted to form an alpha-helix. Gln437 would lie one turn of the alpha-helix away from Thr440, which was previously implicated in recognition of position -12. The rpoD-QH437 mutation described here lends further support to the model that region 2.4 of sigma is involved in recognition of the 5' end of the -10 hexamer. In addition, two rpoD mutations with non-specific effects on promoter recognition are described.

A mutant Escherichia coli sigma 70 subunit of RNA polymerase with altered promoter specificity.

A mutation is described that alters the promoter specificity of sigma 70, the primary sigma factor of Escherichia coli RNA polymerase. In strains carrying both the mutant and wild-type sigma gene (rpoD), the mutant sigma causes a large increase in the activity of mutant P22 ant promoters with A.T or C.G instead of the wild-type, consensus G.C base-pair at position -33, the third position of the consensus -35 hexamer 5'-TTGACA-3'. There is little or no effect on the activities of the wild-type and 23 other mutant ant promoters, including one with T.A at -33. The mutant sigma also activates E. coli lac promoters with A.T or C.G, but not T.A, at the corresponding position. The rpoD mutation (rpoD-RH588) changes a CGT codon to CAT. The corresponding change in sigma 70 is Arg588----His. This residue is in a region that is conserved among most sigma factors, a region that is also homologous with the helix-turn-helix motif of DNA-binding proteins. These results suggest that this region of sigma 70 is directly involved in recognition of the -35 hexamer.

The effects of mutations in the ant promoter of phage P22 depend on context.

Recombination was used to construct 22 two- or three-way combinations of down- and up-mutations in Pant, a strong, near-consensus promoter of phage P22. The relative strengths of these promoters in vivo were assayed by fusing them to an ant/lacZ gene fusion and measuring beta-galactosidase levels produced by lysogens carrying the fusions on single-copy prophages. The results of these assays show that the magnitude of the effect of a promoter mutation can vary considerably when its context is changed by the presence of another mutation. In addition, as Pant approaches conformity with the consensus promoter sequence, the up-mutations decrease promoter strength, even though the same mutations increase promoter strength in the presence of a down-mutation. These context effects imply that individual consensus base pairs cannot be considered to contribute to promoter strength independently.

Control of gene expression in bacteriophage P22 by a small antisense RNA. I. Characterization in vitro of the Psar promoter and the sar RNA transcript.

The characterization in vitro of a newly discovered promoter (Psar) in the bacteriophage P22 immI region is described. Psar is located within the ant gene and is directed toward the major immI promoter, Pant. The entire intercistronic region between the P22 arc and ant genes (69 bp) is transcribed. The initiation and termination of sar (small antisense regulatory) RNA transcription are unusual. Frequent abortive initiation occurs in the presence of all four NTPs; RNA products 3-13 nucleotides in length are produced in about 15- to 25-fold larger numbers than full-length transcripts. Termination of sar RNA synthesis occurs after transcription of the first and second Ts of a TTTA sequence following a region of hyphenated dyad symmetry. The effects of convergent transcription between Pant and Psar were investigated on linear and supercoiled templates. Active transcription from Pant interferes with full-length transcription from Psar; several factors that interfere with Pant initiation (e.g., Pant down-mutation, Mnt repressor protein, Arc repressor protein) result in indirect activation of sar RNA synthesis. The sar RNA pairs rapidly with ant mRNA to form a stable stoichiometric complex. The location and properties of Psar suggest an important regulatory function for sar RNA as a negative effector of ant expression. The results of Wu et al. (this issue) support this suggestion.

Control of gene expression in bacteriophage P22 by a small antisense RNA. II. Characterization of mutants defective in repression.

Phage P22 produces antirepressor protein early after infection from a transcript initiated at the Pant promoter. After the first few minutes of infection, transcription from Pant is repressed by a protein encoded by the arc gene. Antirepressor is not produced late in infection, even though the antirepressor gene, ant, is transcribed from the late operon promoter Plate. We describe the isolation of P22 mutants that synthesize antirepressor from the Plate transcript. The mutations inactivate a promoter Psar, which lies within the ant coding sequence and directs the synthesis of sar RNA, a small antisense regulatory RNA complementary to the ant ribosome binding site. Characterization of the Psar down-mutants shows that transcription from Psar interferes with synthesis of antirepressor from both the Plate and Pant transcripts. Since sar RNA represses synthesis of antirepressor in trans, we propose that sar RNA base-pairs with ant mRNA to inhibit antirepressor synthesis at a post-transcriptional level. The role and importance of sar RNA in P22 biology are discussed.

Structural and regulatory divergence among site-specific recombination genes of lambdoid phage.

The lambdoid bacteriophage phi 80 and P22 have site-specific recombination systems similar to that of lambda. Each of the three phage has a different insertion specificity, but structural analysis of their attachment sites suggests that the three recombination pathways share similar features. In this study, we have identified and sequenced the int and xis genes of phi 80 and P22. phi 80 int and xis were identified using a plasmid recombination assay in vivo, and the P22 genes were mapped using Tn1 insertion mutations. In all three phage, the site-specific recombination genes are located directly adjacent to the phage attachment site. Interestingly, the transcriptional orientation of the phi 80 int gene is opposite to that of lambda and P22 int, resulting in convergent transcription of phi 80 int and xis. Because of its transcriptional orientation, phi 80 int cannot be expressed by the major leftward promoter, PL, and the regulatory strategy of phi 80 integration and excision must differ significantly from that of lambda. The deduced amino acid sequences of the recombination proteins of the three systems show surprisingly little homology. Sequences homologous to the lambda PI promoter are more conserved than the protein-coding sequences. Nevertheless, the Int proteins are locally related in the C-terminal sequences, particularly for a stretch of some 25 amino acid residues that lie approximately 50 residues from the C terminus. The Xis proteins can be aligned at their N termini.

The bacteriophage P22 arc and mnt repressors. Overproduction, purification, and properties.

The arc and mnt genes of bacteriophage P22 encode small repressor proteins. We have cloned these genes onto plasmids that overproduce Arc and Mnt to greater than 1% of the soluble cellular protein. Both proteins were purified to greater than 95% homogeneity, and N-terminal sequences and amino acid compositions were determined. These data, in combination with previously determined gene sequences, establish the complete protein sequences for Arc (53 residues) and Mnt (82 residues). Both proteins have melting temperatures between 45 and 55 degrees C and can be renatured to a fully active species. Arc is a dimer in solution and Mnt is a tetramer.

Mutations that improve the ant promoter of Salmonella phage P22.

Mutations that increase the activity of the promoter for the antirepressor gene of phage P22 were isolated by pseudoreversion of four severe promoter-down mutations. The sequence changes in these pseudorevertants include single base pair substitutions, single base pair deletions, tandem double base pair deletions and multisite mutations. The single base pair substitutions change nonconsensus base pairs to consensus base pairs at positions -14 and -8. The other mutations provide support for the idea that the length of the spacer region between the conserved -35 and -10 hexamers is an important determinant of promoter strength. Deletions of one or two base pairs in the spacer region apparently activate an alternate -10 hexamer by shifting it from a spacing of 19 base pairs to a spacing of 18 or 17 base pairs, respectively.

The phi 80 and P22 attachment sites. Primary structure and interaction with Escherichia coli integration host factor.

Although the lambdoid bacteriophage phi 80 and P22 possess site-specific recombination systems analogous to bacteriophage lambda, they have different attachment (att) site specificities. We have identified and determined the nucleotide sequences of the att sites of phi 80 and P22 and have examined the interaction of these sites with purified Escherichia coli integration host factor (IHF). The sizes of the homologous core regions of the att sites vary greatly: P22 has a 46-base pair core, while phi 80 and lambda have 17- and 15-base pair cores, respectively. The core sequences of the three phage show no significant homology, although dispersed regions of homology in arm sequences indicate that the three phage att sites are related. All three att sites have a high A + T composition, and restriction fragments carrying these sites migrate anomalously upon polyacrylamide gel electrophoresis. IHF binds to a site to the left of the common core in the phi 80 and P22 phage att sites (attP) and to a site to the right of the core in P22 attP and attB (the bacterial att site). In the lambda system, IHF interacts with three regions on attP (designated H1, H2, and H') and none on attB (Craig N., and Nash, H.A. (1984) Cell 39, 707-716). Alignment of the IHF sites of all three phage results in a consensus sequence for IHF binding, Pyr-AANNNNTTGATAT. Among the three phage, the number of IHF sites differs; however, the location and orientation of the binding sites in relation to the respective core regions are well conserved. An IHF site analogous to lambda H2 is present in both phi 80 and P22 attP, while a site analogous to lambda H' is present in P22 attP. This conservation suggests that IHF plays a very similar role in the site-specific recombination pathways of all three phage, and that the flanking arm sequences are necessary for phi 80 and P22 attP function, as is the case for lambda attP function. These structural similarities presumably reflect a conservation of the mechanism of site-specific recombination for the three phage.

Changing the DNA-binding specificity of a repressor.

The Mnt repressor of Salmonella phage P22 recognizes a 17 base pair operator. We constructed an Mnt binding site with two symmetric operator-constitutive mutations. We then selected Mnt mutants that have lost the ability to bind the wild-type operator but acquired the ability to bind the mutant operator. Four independent mutations change the same CAC codon in the mnt gene to CCT or CCC. The corresponding amino acid change in Mnt is His6 leads to Pro. DNA binding assays with purified wild-type and mutant proteins confirm the change of binding specificity in vitro. Wild-type Mnt binds strongly to the wild-type operator, but binds the mutant operator with 1000-fold less affinity. The mutant Mnt binds with converse affinities to the two operators.

Primary structure of the immI immunity region of bacteriophage P22.

The DNA sequence of the immI immunity region of bacteriophage P22 has been determined. This region includes the ant gene, which encodes the P22 antirepressor protein, and the mnt and arc genes, which encode proteins that negatively regulate antirepressor synthesis. We have purified antirepressor protein and selected tryptic peptides of antirepressor, and have determined the amino terminal sequences and amino acid composition of these molecules. These data, in combination with the DNA sequence, locate the ant gene and define the complete amino acid sequence of antirepressor (300 residues). The mnt and arc genes have been located by sequencing the mnt-am343 and arc-amH1605 mutations. The Mnt and Arc proteins are predicted to be small, basic polypeptides that are homologous in amino acid sequence. The Mnt protein also shows significant sequence homology with the lambda Cro protein. The arc and ant genes are transcribed rightward from the Pant promoter, while mnt is transcribed leftward from a promotor that may overlap Pant. The Mnt protein apparently acts by binding to an operator site located immediately adjacent to the startpoint of Pant transcription.