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.

Recombineering: a powerful new tool for mouse functional genomics.

Highly efficient phage-based Escherichia coli homologous recombination systems have recently been developed that enable genomic DNA in bacterial artificial chromosomes to be modified and subcloned, without the need for restriction enzymes or DNA ligases. This new form of chromosome engineering, termed recombinogenic engineering or recombineering, is efficient and greatly decreases the time it takes to create transgenic mouse models by traditional means. Recombineering also facilitates many kinds of genomic experiment that have otherwise been difficult to carry out, and should enhance functional genomic studies by providing better mouse models and a more refined genetic analysis of the mouse genome.

High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides.

Homologous DNA recombination is a fundamental, regenerative process within living organisms. However, in most organisms, homologous recombination is a rare event, requiring a complex set of reactions and extensive homology. We demonstrate in this paper that Beta protein of phage lambda generates recombinants in chromosomal DNA by using synthetic single-stranded DNAs (ssDNA) as short as 30 bases long. This ssDNA recombination can be used to mutagenize or repair the chromosome with efficiencies that generate up to 6% recombinants among treated cells. Mechanistically, it appears that Beta protein, a Rad52-like protein, binds and anneals the ssDNA donor to a complementary single-strand near the DNA replication fork to generate the recombinant. This type of homologous recombination with ssDNA provides new avenues for studying and modifying genomes ranging from bacterial pathogens to eukaryotes. Beta protein and ssDNA may prove generally applicable for repairing DNA in many organisms.

A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA.

Recently, a highly efficient recombination system for chromosome engineering in Escherichia coli was described that uses a defective lambda prophage to supply functions that protect and recombine a linear DNA targeting cassette with its substrate sequence (Yu et al., 2000, Proc. Natl. Acad. Sci. USA 97, 5978-5983). Importantly, the recombination is proficient with DNA homologies as short as 30-50 bp, making it possible to use PCR-amplified fragments as the targeting cassette. Here, we adapt this prophage system for use in bacterial artificial chromosome (BAC) engineering by transferring it to DH10B cells, a BAC host strain. In addition, arabinose inducible cre and flpe genes are introduced into these cells to facilitate BAC modification using loxP and FRT sites. Next, we demonstrate the utility of this recombination system by using it to target cre to the 3' end of the mouse neuron-specific enolase (Eno2) gene carried on a 250-kb BAC, which made it possible to generate BAC transgenic mice that specifically express Cre in all mature neurons. In addition, we show that fragments as large as 80 kb can be subcloned from BACs by gap repair using this recombination system, obviating the need for restriction enzymes or DNA ligases. Finally, we show that BACs can be modified with this recombination system in the absence of drug selection. The ability to modify or subclone large fragments of genomic DNA with precision should facilitate many kinds of genomic experiments that were difficult or impossible to perform previously and aid in studies of gene function in the postgenomic era.

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.

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.

Rapid engineering of bacterial artificial chromosomes using oligonucleotides.

A rapid method obviating the use of selectable markers to genetically manipulate large DNA inserts cloned into bacterial artificial chromosomes is described. Mutations such as single-base changes, deletions, and insertions can be recombined into a BAC by using synthetic single-stranded oligonucleotides as targeting vectors. The oligonucleotides include the mutated sequence flanked by short homology arms of 35-70 bases on either side that recombine with the BAC. In the absence of any selectable marker, modified BACs are identified by specific PCR amplification of the mutated BAC from cultures of pooled bacterial cells. Each pool represents about 10 electroporated cells from the original recombination mixture. Subsequently, individual clones containing the desired alteration are identified from the positive pools. Using this BAC modification method, we have observed a frequency of one recombinant clone per 90-260 electroporated cells. The combination of high targeting frequency and the sensitive yet selective PCR-based screening method makes BAC manipulation using oligonucleotides both rapid and simple.

Isolation and preliminary characterization of the human and mouse homologues of the bacterial cell cycle gene era.

Era is an essential GTPase that is required for proper cell cycle progression and cell division in Escherichia coli and is found in nearly all bacteria sequenced to date. To determine whether Era is also present in eukaryotic organisms, we searched the dbEST database and found EST clones coding for proteins that were similar to Era. Full sequencing of these ESTs from human and mouse identified a conserved homologue, ERAL1 (Era-like 1). ERAL1 maps to 17q11.2 in human and is located in the syntenic region of mouse chromosome 11. ERAL1 may be an attractive candidate for a tumor suppressor gene since ERAL1 is located in a chromosomal region where loss of heterozygosity is often associated with various types of cancer.

The structure of the transcriptional antiterminator NusB from Escherichia coli.

We have determined the solution structure of NusB, a transcription antitermination protein from Escherichia coli. The structure reveals a novel, all alpha-helical protein fold. NusB mutations that cause a loss of function (NusB5) or alter specificity for RNA targets (NusB101) are localized to surface residues and likely affect RNA-protein or protein-protein interactions. Residues that are highly conserved among homologs stabilize the protein core. The solution structure of E. coli NusB presented here resembles that of Mycobacterium tuberculosis NusB determined by X-ray diffraction, but differs substantially from a solution structure of E. coli NusB reported earlier.

An efficient recombination system for chromosome engineering in Escherichia coli.

A recombination system has been developed for efficient chromosome engineering in Escherichia coli by using electroporated linear DNA. A defective lambda prophage supplies functions that protect and recombine an electroporated linear DNA substrate in the bacterial cell. The use of recombination eliminates the requirement for standard cloning as all novel joints are engineered by chemical synthesis in vitro and the linear DNA is efficiently recombined into place in vivo. The technology and manipulations required are simple and straightforward. A temperature-dependent repressor tightly controls prophage expression, and, thus, recombination functions can be transiently supplied by shifting cultures to 42 degrees C for 15 min. The efficient prophage recombination system does not require host RecA function and depends primarily on Exo, Beta, and Gam functions expressed from the defective lambda prophage. The defective prophage can be moved to other strains and can be easily removed from any strain. Gene disruptions and modifications of both the bacterial chromosome and bacterial plasmids are possible. This system will be especially useful for the engineering of large bacterial plasmids such as those from bacterial artificial chromosome libraries.

Crystal structure of ERA: a GTPase-dependent cell cycle regulator containing an RNA binding motif.

ERA forms a unique family of GTPase. It is widely conserved and essential in bacteria. ERA functions in cell cycle control by coupling cell division with growth rate. ERA homologues also are found in eukaryotes. Here we report the crystal structure of ERA from Escherichia coli. The structure has been determined at 2.4-A resolution. It reveals a two-domain arrangement of the molecule: an N-terminal domain that resembles p21 Ras and a C-terminal domain that is unique. Structure-based topological search of the C domain fails to reveal any meaningful match, although sequence analysis suggests that it contains a KH domain. KH domains are RNA binding motifs that usually occur in tandem repeats and exhibit low sequence similarity except for the well-conserved segment VIGxxGxxIK. We have identified a betaalphaalphabeta fold that contains the VIGxxGxxIK sequence and is shared by the C domain of ERA and the KH domain. We propose that this betaalphaalphabeta fold is the RNA binding motif, the minimum structural requirement for RNA binding. ERA dimerizes in crystal. The dimer formation involves a significantly distorted switch II region, which may shed light on how ERA protein regulates downstream events.

Escherichia coli nusG mutations that block transcription termination by coliphage HK022 Nun protein.

The Escherichia coli nusG gene product is required for transcription termination by phage HK022 Nun protein at the lambda nutR site in vivo. We show that it is also essential for Nun termination at lambda nutL. Three recessive mis-sense nusG mutations have been isolated that inhibit termination by Nun at lambda nutR. The mutations are ineffective in a lambda pL nutL fusion, even when lambda nutR replaces lambda nutL. The mutant strains support lambda growth, indicating that lambda N antitermination activity is not impaired. Transcription arrest by Nun in vitro is stimulated by NusG protein at both lambda nutR and lambda nutL. Mutant NusG protein fails to enhance transcriptional arrest by Nun at either site. The mutant protein, like the wild-type protein, suppresses transcriptional pausing by RNA polymerase and stimulates Rho-dependent termination. These results imply that the role of NusG in Nun termination may be distinct from its roles in other transcription reactions.

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.

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.

Genetic uncoupling of the dsRNA-binding and RNA cleavage activities of the Escherichia coli endoribonuclease RNase III--the effect of dsRNA binding on gene expression.

RNase III, a double-stranded RNA-specific endonuclease, is proposed to be one of Escherichia coli's global regulators because of its ability to affect the expression of a large number of unrelated genes by influencing post-transcriptional control of mRNA stability or mRNA translational efficiency. Here, we describe the phenotypes of bacteria carrying point mutations in rnc, the gene encoding RNase III. The substrate recognition and RNA-processing properties of mutant proteins were analysed in vivo by measuring expression from known RNase III-modulated genes and in vitro from the proteins' binding and cleavage activities on known double-stranded RNA substrates. Our results show that although the point mutation rnc70 exhibited all the usual rnc null-like phenotypes, unlike other mutations, it was dominant over the wild-type allele. Multicopy expression of rnc70 could suppress a lethal phenotype of the wild-type rnc allele in a certain genetic background; it could also inhibit the RNase III-mediated activation of lambdaN gene translation by competing for the RNA-binding site of the wild-type endonuclease. The mutant protein failed to cleave the standard RNase III substrates in vitro but exhibited an affinity for double-stranded RNA when passed through poly(rI):poly(rC) columns. Filter binding and gel-shift assays with purified Rnc70 showed that the mutant protein binds to known RNase III mRNA substrates in a site-specific manner. In vitro processing reactions with purified enzyme and labelled RNA showed that the in vivo dominant effect of the mutant enzyme over the wild-type was not necessarily caused by formation of mixed dimers. Thus, the rnc70 mutation generates a mutant RNase III with impaired endonucleolytic activity but without blocking its ability to recognize and bind double-stranded RNA substrates.

Cell cycle arrest in Era GTPase mutants: a potential growth rate-regulated checkpoint in Escherichia coli.

Era is a low-molecular-weight GTPase essential for Escherichia coli viability. The gene encoding Era is found in the rnc operon, and the synthesis of both RNase III and Era increases with growth rate. Mutants that are partially defective in Era GTPase activity or that are reduced in the synthesis of wild-type Era become arrested in the cell cycle at the predivisional two-cell stage. The partially defective Era GTPase mutation (era1) suppresses several temperature-sensitive lethal alleles that affect chromosome replication and chromosome partitioning but not cell division. Our results suggest that Era plays an important role in cell cycle progression at a specific point in the cycle, after chromosome partitioning but before cytokinesis. Possible functions for Era in cell cycle progression and the initiation of cell division are discussed.

Control of ftsZ expression, cell division, and glutamine metabolism in Luria-Bertani medium by the alarmone ppGpp in Escherichia coli.

Inactivation of transcription factor sigma54, encoded by rpoN (glnF), restores high-temperature growth in Luria-Bertani (LB) medium to strains containing the heat-sensitive cell division mutation ftsZ84. Mutational defects in three other genes involved in general nitrogen control (glnD, glnG, and glnL) also suppress lethal filamentation. Since addition of glutamine to LB medium fully blocks suppression by each mutation, the underlying cause of suppression likely derives from a stringent response to the limitation of glutamine. This model is supported by several observations. The glnL mutation requires RelA-directed synthesis of the nutrient alarmone ppGpp to suppress filamentation. Artificially elevated levels of ppGpp suppress ftsZ84, as do RNA polymerase mutations that reproduce global effects of the ppGpp-induced state. Both the glnF null mutation and an elevated copy number of the relA gene similarly affect transcription from the upstream (pQ) promoters of the ftsQAZ operon, and both of these genetic conditions increase the steady-state level of the FtsZ84 protein. Physiological suppression of ftsZ84 by a high salt concentration was also shown to involve RelA. Additionally, we found that the growth of a glnF or glnD strain on LB medium depends on RelA or supplemental glutamine in the absence of RelA function. These data expand the roles for ppGpp in the regulation of glutamine metabolism and the expression of FtsZ during cell division.

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.

Translational repression by a transcriptional elongation factor.

One of the classical positive regulators of gene expression is bacteriophage lambda N protein. N regulates the transcription of early phage genes by participating in the formation of a highly processive, terminator-resistant transcription complex and thereby stimulates the expression of genes lying downstream of transcriptional terminators. Also included in this antiterminating transcription complex are an RNA site (NUT) and host proteins (Nus). Here we demonstrate that N has an additional, hitherto unknown regulatory role, as a repressor of the translation of its own gene. N-dependent repression does not occur when NUT is deleted, demonstrating that N-mediated antitermination and translational repression both require the same cis-acting site in the RNA. In addition, we have identified one nut and several host mutations that eliminate antitermination and not translational repression, suggesting the independence of these two N-mediated mechanisms. Finally, the position of nutL with respect to the gene whose expression is repressed is important.

Characterization of mutations affecting the Escherichia coli essential GTPase era that suppress two temperature-sensitive dnaG alleles.

Two suppressor mutations of the temperature-sensitive DNA primase mutant dnaG2903 have been characterized. The gene responsible for suppression, era, encodes an essential GTPase of Escherichia coli. One mutation, rnc-15, is an insertion of an IS1 element within the leader region of the rnc operon and causes a polar defect on the downstream genes of the operon. A previously described polar mutation, rnc-40, was also able to suppress dnaG2903. The other mutation, era-1, causes a single amino acid substitution (P17R) in the G1 region of the GTP-binding domain of Era. Analysis of the GTPase activity of the Era-1 mutant protein showed a four- to five-fold decrease in the ability to convert GTP to GDP. Thus, lowered expression of wild-type Era caused by the polar mutations and reduced GTPase activity caused by the era-1 mutation suppresses dnaG2903 as well as a second dnaG allele, parB. Phenotypic analysis of the era-1 mutant at 25 degrees C showed that 10% of the cells contain four segregated nucleoids, indicative of a delay in cell division. Possible mechanisms of suppression of dnaG and roles for Era are discussed.