Functional importance of sequence in the stem-loop of a transcription terminator.

Intrinsic transcription terminators of prokaryotes are distinguished by a common RNA motif: a stem-loop structure high in guanine and cytosine content, followed by multiple uridine residues. Models explaining intrinsic terminators postulate that the stem-loop sequence is necessary only to form structure. In the tR2 terminator of coliphage lambda, single-nucleotide changes reducing potential RNA stem stability eliminated tR2 activity, and a compensatory change that restored the stem structure restored terminator activity. However, multiple changes in the stem sequence that should have either maintained or increased stability reduced terminator activity. These results suggest that the ability of the stem-loop structure to signal transcription termination depends on sequence specificity and secondary structure.

Bacteriophage lambda: alive and well and still doing its thing.

The lambda (lambda) family of bacteriophages continues to provide significant insights into the understanding of basic biological processes, as well as useful technological innovations. Areas in which recent advances have occurred include transcription elongation, repressor interactions, genomics and post-transcriptional regulation. The homologous lambda recombination functions have been exploited as an efficient in vivo recombinant engineering system for functional genomic studies. The virulence of some pathogenic strains of Escherichia coli is enhanced by the expression of Shiga toxin (stx) genes encoded on a resident lambdoid prophage. Recent work suggests that the phage regulatory network may be a significant contributor to toxin production and release by these pathogenic E. coli.

Crystallographic and modeling studies of RNase III suggest a mechanism for double-stranded RNA cleavage.

BACKGROUND: Aquifex aeolicus Ribonuclease III (Aa-RNase III) belongs to the family of Mg(2+)-dependent endonucleases that show specificity for double-stranded RNA (dsRNA). RNase III is conserved in all known bacteria and eukaryotes and has 1-2 copies of a 9-residue consensus sequence, known as the RNase III signature motif. The bacterial RNase III proteins are the simplest, consisting of two domains: an N-terminal endonuclease domain, followed by a double-stranded RNA binding domain (dsRBD). The three-dimensional structure of the dsRBD in Escherichia coli RNase III has been elucidated; no structural information is available for the endonuclease domain of any RNase III. RESULTS: We present the crystal structures of the Aa-RNase III endonuclease domain in its ligand-free form and in complex with Mn(2+). The structures reveal a novel protein fold and suggest a mechanism for dsRNA cleavage. On the basis of structural, genetic, and biological data, we have constructed a hypothetical model of Aa-RNase III in complex with dsRNA and Mg(2+) ion, which provides the first glimpse of RNase III in action. CONCLUSIONS: The functional Aa-RNase III dimer is formed via mainly hydrophobic interactions, including a "ball-and-socket" junction that ensures accurate alignment of the two monomers. The fold of the polypeptide chain and its dimerization create a valley with two compound active centers at each end of the valley. The valley can accommodate a dsRNA substrate. Mn(2+) binding has significant impact on crystal packing, intermolecular interactions, thermal stability, and the formation of two RNA-cutting sites within each compound active center.

Regulatory defects of a conditionally lethal nusAts mutant of Escherichia coli. Positive and negative modulator roles of NusA protein in vivo.

Previous studies have attributed two activities to the NusA protein of Escherichia coli; namely, termination and antitermination of transcription. To examine these activities, we isolated a temperature-sensitive mutant of the nusA gene (nusAts11). The mutant cells produce a thermolabile NusA protein and grow at 32 degrees C, but not at 42 degrees C. At 42 degrees C, nusAts11 is recessive to nusA+ and nusA1, indicating the absence of its active gene product at that temperature. In the mutant, the efficiency of termination at the lambda tR1 terminator decreases, resulting in an increased expression of distal gene(s). On the other hand, the synthesis of the beta-galactosidase and beta beta' subunits of RNA polymerase is reduced in the mutant. This mimics effects seen in vitro when NusA protein is removed from a coupled transcription-translation system. These results suggest that the NusA protein plays both negative and positive modulator roles in vivo. The mutation nusAts11, unlike nusA1, does not block lambda phage growth at non-permissive temperatures, suggesting that NusA protein is not required for N antitermination in the mutant. Besides, the nusAts11 allows lambda Nam7Nam53byp phage growth under sup0 conditions, indicating that the N antitermination function is dispensable (at least partly) in this mutant.

Mutations of bacteriophage lambda that define independent but overlapping RNA processing and transcription termination sites.

Bacteriophage lambda int gene expression is regulated differentially from transcripts originated at the pL and pI promoters. Transcripts initiated at pI terminate at the site tI and express int gene product efficiently. Polymerases starting at pL do not terminate at tI, due to the antiterminating activity of lambda N protein. The pL transcripts are unable to express Int protein efficiently because sib, a control site overlapping tI in the unterminated RNA, is processed by host RNase III. We have isolated lambda sib- mutants by their inability to inhibit int expression from pL transcripts. sib mutations were genetically mapped to the left of the lambda attachment site, and do not structurally alter this site for recombination. Several sib mutations do alter the nucleotide sequence of the overlapping sib and tI sites. The lambda sib- mutants tested prevent RNA processing but do not affect transcription termination in vivo.

Bacteriophage lambda N-dependent transcription antitermination. Competition for an RNA site may regulate antitermination.

Bacteriophage lambda controls the expression of its early genes in a temporal manner by a series of transcription termination and antitermination events. This antitermination requires the lambda N protein as well as host proteins called Nus, and cis-acting sites called nut. Following transcription of the nut site, N and Nus proteins bind to the nut RNA and modify the transcription complex to a termination-resistant form. The nut site is a composite of at least two components; one is the boxB hairpin structure which interacts with N. The other is boxA, a nine-nucleotide sequence upstream of boxB. To understand more about the formation of the antitermination complex, we have characterized the effect of point mutations in and deletions of boxA on antitermination. Point mutations in boxA were found to either enhance or reduce N-mediated antitermination. Several boxA deletions, on the other hand, had little effect on antitermination other than to eliminate the requirement for the NusB host protein. To explain these observations, we propose that at least two factors compete to interact with boxA, NusB and an inhibitor of the antitermination reaction. In addition, we propose that NusB is required to prevent the inhibitor from binding at boxA. The results with various nusB and boxA mutations can be explained by this model of competition between NusB and an inhibitor for boxA RNA.

Isolation of plaque-forming, galactose-transducing strains of phage lambda.

Plaque-forming, galactose-transducing lambda strains have been isolated from lysogens in which bacterial genes have been removed from between the galactose operon and the prophage by deletion mutation.-A second class has been isolated starting with a lysogenic strain which carries a deletion of the genes to the right of the galactose operon and part of the prophage. This strain was lysogenized with a second lambda phage to yield a lysogen from which galactose-transducing, plaque-forming phages were obtained. These plaque-forming phages were found to be genetically unstable, due to a duplication of part of the lambda chromosome. The genetic instability of these partial diploid strains is due to homologous genetic recombindation between the two identical copies of the phage DNA comprising the duplication. The galactose operon and the duplication of phage DNA carried by these strains is located between the phage lambda P and Q genes.

Transcription termination signals in the nin region of bacteriophage lambda: identification of Rho-dependent termination regions.

The approximately 3-kb nin region of bacteriophage lambda, located between genes P and Q contains transcription termination signals as well as 10 open reading frames. Deletions in the nin region frees phage growth from dependence on the lambda-encoded N-transcription antitermination system, conferring a Nin phenotype (N-independence). A subregion of nin, roc, is defined by a 1.9-kb deletion (delta roc) which partially frees lambda growth from the requirement for N antitermination. The roc region has strong transcription termination activity as assayed by a plasmid-based terminator testing system. We report the following features of the roc region: the biologically significant terminators in the roc region are Rho dependent, deletion analysis located the biologically significant termination signals to a 1.2 kb-segment of roc, and analysis of other deletions and point mutations in the roc region suggested at least two biologically significant regions of termination, tR3 (extending from bp 42020 to 42231) and tR4 (extending from bp 42630 to 42825).

A new system to place single copies of genes, sites and lacZ fusions on the Escherichia coli chromosome.

To place a single-copy lacZ fusion on the E. coli chromosome, a method was developed based on in vivo homologous DNA recombination through P1 transduction. The fusions, initially constructed on plasmids, are crossed to lambdalacZ fusion vectors which are then lysogenized at the chromosomal lambda att site. The features of the new system are: (1) lambda lysogens carrying the fusion are made without regard for copy number; (2) P1 transduction from the lysogenic strain into an appropriate recipient generates the single-copy fusion; (3) The lacZ fusion has no prophage associated with it; (4) the lacZ fusion can be transferred by P1 transduction to other strains, simply by selecting for an antibiotic marker; (5) the system can be widely applied to construct single copies of any gene or site placed between bla and lacZ on the standard lacZ fusion plasmid vectors; and (6) the single-copy construct flanked by prophage att sites can be excised by site-specific recombination to generate non-replicating circular DNA of the clone or a cell cured of the construct.

Cloning, sequencing and expression of the dnaJ gene of Coxiella burnetii.

A 6-kb EcoRI genomic DNA fragment of Coxiella burnetii, isolated from a recombinant bacteriophage lambda ZapII library, allowed heterologous genetic complementation of Escherichia coli deleted for its dnaJ gene. The C. burnetii dnaJ gene was expressed in E. coli and identified by Western blot analysis using polyclonal antibodies raised against purified E. coli DnaJ protein. Deletion mapping and genetic complementation demonstrated that C. burnetii dnaJ is present on a 2-kb EcoRI-HindIII genomic DNA fragment, from which the nt sequence of the C. burnetii dnaJ gene was determined.

Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans.

Genetic interference mediated by double-stranded RNA (RNAi) has been a valuable tool in the analysis of gene function in Caenorhabditis elegans. Here we report an efficient induction of RNAi using bacteria to deliver double-stranded RNA. This method makes use of bacteria that are deficient in RNaseIII, an enzyme that normally degrades a majority of dsRNAs in the bacterial cell. Bacteria deficient for RNaseIII were engineered to produce high quantities of specific dsRNA segments. When fed to C. elegans, such engineered bacteria were found to produce populations of RNAi-affected animals with phenotypes that were comparable in expressivity to the corresponding loss-of-function mutants. We found the method to be most effective in inducing RNAi for non-neuronal tissue of late larval and adult hermaphrodites, with decreased effectiveness in the nervous system, in early larval stages, and in males. Bacteria-induced RNAi phenotypes could be maintained over the course of several generations with continuous feeding, allowing for convenient assessments of the biological consequences of specific genetic interference and of continuous exposure to dsRNAs.

Improved bacterial hosts for regulated expression of genes from lambda pL plasmid vectors.

The construction and use of a set of Escherichia coli strains with defective lambda prophages that facilitate expression of genes cloned in lambda pL-plasmid vectors is described. These bacteria allow high and regulated expression of such genes, whereas a kanamycin-resistance marker (KmR) on the prophage allows easy identification and genetic transfer from strain to strain. Optimal conditions for examining gene expression with the pL-vector systems using these strains are discussed.

Analysis of the DnaK molecular chaperone system of Francisella tularensis.

We have cloned the Francisella tularensis (Ft) grpE-dnaK-dnaJ heat-shock genes which are organized in that order. These genes allow heterologous genetic complementation of each respective mutant strain of Escherichia coli (Ec) for bacteriophage lambda growth. The nucleotide sequences of the Ft grpE-dnaK-dnaJ genes and the deduced amino-acid sequences share significant homologies with their respective Ec counterparts. The Ft DnaK and DnaJ proteins cross-react with polyclonal antibodies raised against the respective Ec proteins. The grpE-dnaK-dnaJ genes of Ft are organized in a fashion that is more characteristic of Gram+ bacteria.

A Francisella tularensis DNA clone complements Escherichia coli defective for the production of Era, an essential Ras-like GTP-binding protein.

We cloned the era gene of Francisella tularensis from a plasmid library by heterologous genetic complementation of an Escherichia coli mutant conditionally defective for the production of Era, an essential protein for cell growth. Nucleotide sequence analysis indicated that, in F. tularensis, era constitutes a single gene operon. ORFs aspC and mdh encoding aspartate aminotransferase and malate dehydrogenase, respectively, flank era in F. tularensis. Although classified as Gram-, the flanking regions and the relative location of era in F. tularensis are distinctly different from those of typical Gram- and Gram+ bacteria. Computer analysis of bacterial Era protein sequences identified conserved domains in addition to the common G domains of most GTP-binding proteins.

Detection of heterologous fusion proteins in Escherichia coli with a monoclonal antibody.

Several laboratories have constructed expression vectors for the production of heterologous fusion proteins containing the N-terminal 13 amino acids of the bacteriophage lambda cII-coded protein in Escherichia coli. We have prepared a monoclonal antibody to a synthetic peptide having this CII amino acid sequence and have found that this antibody reacts with authentic CII protein in Western blot tests and with most CII peptide-containing fusion proteins in both radioimmunoprecipitation and Western blot assays. However, there are some CII-hybrid protein species with which the antibody does not react. Our findings indicate that this antibody is a valuable tool for detecting and purifying expressed proteins and in studying their structure and function.

Murine monoclonal antibodies which recognize active sites of Escherichia coli NusA protein and epitope mapping by gene fusion.

Seven hybridomas producing murine monoclonal antibodies reactive against NusA protein of Escherichia coli were prepared. Antigenic determinants of these monoclonal antibodies have been mapped by immunoblotting analyses using fusion proteins containing parts of NusA. The epitope of the N14 antibody maps in a hydrophobic amino acid (aa) cluster and consists of at least Ala-181 and Ser-183 residues. nusA1 and nusA11 mutations, which cause aa changes of these residues, abolish the antigenic reactivity to the N14 antibody. These antibodies react with intact NusA protein, indicating that the epitopes are exposed on the surface of NusA. Most of these epitopes cluster around the nusA1 and nusA11 mutation loci.

A plasmid vector for cloning and expression of gene segments: expression of an HTLV-I envelope gene segment.

We describe a cloning-expression vector system for selecting DNA fragments containing open reading frames (ORFs) and expressing them as beta-galactosidase (beta Gal) hybrid fusion proteins. The plasmid vector, pWS50, utilizes the very strong and easily regulated bacteriophage lambda promoter pL, and the efficient translation initiation signals of the N-terminal segment of the lambda cII gene. Fused distally to and out of translational phase with cII is the E. coli lacZ gene, lacking its own transcriptional and translational initiating signals. A unique restriction enzyme site (NruI) is located between the upstream regulatory sequences and the lacZ gene, which provides a cloning site for the insertion of blunt ended DNA fragments. In addition, there are two other unique restriction sites (NheI and BamHI) located in this region which can also be used as closing sites. If a DNA fragment does not contain any translation termination codons (i.e., an ORF), and is inserted correctly into the vector, the translational reading frame between cII and lacZ can be restored. Colonies containing these recombinants can be easily screened as LacZ+ on lactose indicator media. The beta-galactosidase fusion proteins produced from the LacZ+ recombinants are identified on sodium dodecyl sulfate polyacrylamide gels by their large size and high level of production. To test the ORF cloning-expression system, a segment of the human T-cell lymphotrophic virus type I envelope gene was cloned and expressed at high levels. The envelope-beta Gal fusion protein was recognized by antibodies in serum from a patient with adult T-cell leukemia.

Transposition studies of mini-Mu plasmids constructed from the chemically synthesized ends of bacteriophage Mu.

We describe below the chemical synthesis of the right and left ends of bacteriophage Mu and characterize the activity of these synthetic ends in mini-Mu transposition. Mini-Mu plasmids were constructed which carry the synthetic Mu ends together with the Mu A and B genes under control of the bacteriophage lambda pL promoter. Derepression of pL leads to a high frequency of mini-Mu transposition (5.6 X 10(-2) which is dependent on the presence of the Mu ends and the Mu A and B proteins. Five deletion mutants in the Mu ends were tested in the mini-Mu transposition system and their effects on transposition are described.

Purification, characterization and crystallization of ERA, an essential GTPase from Escherichia coli.

ERA is an essential GTPase widely conserved in bacteria. Homologues of ERA are also present in higher eukaryotic cells. ERA is involved in bacterial cell cycle control at a point preceding cell division. In order to aid the functional investigation of ERA and to facilitate structure-function studies, we have undertaken the X-ray crystallographic analysis of this protein. Here, we report the purification and crystallization procedures and results. The purified ERA exhibits nucleotide-binding activity and GTP-hydrolytic activity. ERA is one of the very few multi-domain GTPases crystallized to date.

Sequential assignments and secondary structure of the RNA-binding transcriptional regulator NusB.

The NusB protein is involved in transcriptional regulation in bacteriophage lambda. NusB binds to the RNA form of the nut site and along with N, NusA, NusE and NusG, stabilizes the RNA polymerase transcription complex and allows stable, persistent antitermination. NusB contains a 10 residue Arg-rich RNA-binding motif (ARM) at the N-terminus but is not sequentially homologous to any other proteins. In contrast to other known ARM-containing proteins, NusB forms a stable structure in solution in the absence of RNA. NMR spectroscopy was used to determine that NusB contains six alpha-helices: R10-Q21, 127-F34, V45-L65, Q79-S93, Y100-F114 and D118-L127. The structure of NusB makes it a member of a newly emerging class of alpha-helical RNA-binding proteins.