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J P Richardson

Publications and source records attributed to J P Richardson.

At least 19 recordsLinked to original sources
PubMed2001

Mutational changes of conserved residues in the Q-loop region of transcription factor Rho greatly reduce secondary site RNA-binding.

Transcription factor Rho of Eschericia coli is a ring-shaped homohexameric protein that terminates transcripts by its action on nascent RNAs. To test the functional importance of the phylogenetically highly conserved residues of the Q-loop region, four mutant Rho proteins, S281A, K283A, T286A and D290A, were isolated and analyzed for their biochemical properties. All four proteins were very defective in terminating transcripts in vitro at the bacteriophage lambda tR1 terminator and had corresponding defects in ATP hydrolysis activated by lambda cro RNA. Although the four proteins were normal or near normal in their sensitivity to cleavage with H(2)O(2) in the presence of Fe-EDTA and in their ability to bind to lambda cro RNA and ATP, they were defective in RNA-specific, secondary site interactions. This was indicated by the lack of protection from cleavage at their Q-loops by oligo(C) in the presence of poly(dC), and their defects in ATP hydrolysis activated by oligo(C) in the presence of poly(dC). This evidence, together with the observations that cleavage of the Q-loop residues is protected specifically by RNA, suggests that the Q-loop makes interactions with RNA that are essential for activation of ATP hydrolysis and the termination of transcription.

Adenosine Triphosphatases↗
PubMed2001

Phenotypic characterization of a comprehensive set of bicyclomycin-resistant mutants.

A comprehensive set of bicyclomycin-resistant mutants of transcription termination protein Rho has been characterized in Escherichia coli by in vivo and in vitro assays. Several of the mutant Rho proteins have functional defects. Strains with either the L208R or the S266A mutation in the bacterial chromosome have a higher intracellular concentration of the Rho protein than strains containing a wild-type copy of the rho gene. Strains carrying the L187R, L208R or S266A mutations in the chromosome also have a mutant phenotype; a plasmid-located arabinose promoter is constitutively de-repressed in these strains. The L208R and S266A mutant strains also have a rate of growth defect. When the S266A mutation is located on a high-copy plasmid, the mutant grows more slowly than a wild-type strain. In contrast to the majority of the bicyclomycin-resistant mutants, these two mutants show clear phenotypic differences from wild-type cells. These differences are also seen in vitro. In vitro transcription termination by RhoL208R and RhoS266A is defective at the lambda tR1 terminator, but can be enhanced by NusG. These functionally defective Rho mutations have been located near the putative catalytic site on a model of Rho based on the F1-ATPase. This indicates that this region of the Rho molecule is crucial for Rho function. The crucial region overlaps the putative bicyclomycin-binding site, suggesting an explanation for the efficacy of bicyclomycin as an antibiotic.

Anti-Bacterial Agents↗
PubMed2001

Identification of an RNA-binding Site in the ATP binding domain of Escherichia coli Rho by H2O2/Fe-EDTA cleavage protection studies.

Transcription factor Rho is a ring-shaped, homohexameric protein that causes transcript termination through actions on nascent RNAs that are coupled to ATP hydrolysis. The Rho polypeptide has a distinct RNA binding domain of known structure as well as an ATP binding domain for which a structure has been proposed based on homology modeling. Treatment of Rho with H2O2 in the presence of Fe-EDTA caused single-cut cleavage at a number of points that coincide with solvent-exposed loops in both the known and predicted structures, thereby providing support for the validity of the tertiary and quaternary structural models of Rho. The binding of ATP caused one distinct change in the cleavage pattern, a strong protection at a cleavage point in the P-loop of the ATP binding domain. Binding of RNA and single-stranded DNA (poly(dC)) caused strong protection at several accessible parts of the oligosaccharide/oligonucleotide binding (OB) fold in the RNA binding domain. RNA molecules but not DNA molecules also caused a strong, ATP-dependent protection at a cleavage site in the predicted Q-loop of the ATP binding domain. These results suggest that Rho has two distinct binding sites for RNA. Besides the site composed of multiples of the RNA binding domain, to which single-stranded DNA as well as RNA can bind, it has a separate, RNA-specific site on the Q-loop in the ATP binding domain. In the proposed quaternary structure of Rho, the Q-loops from the six subunits form the upper entrance to the hole in the ring-shaped hexamer through which the nascent transcript is translocated by actions coupled to ATP hydrolyses.

Adenine↗
PubMed2001

Transcription factor Rho does not require a free end to act as an RNA-DNA helicase on an RNA.

Escherichia coli Rho factor is a ring-shaped, homohexameric protein that terminates synthesis of RNA through interactions with the nascent RNA transcript. Because its mechanism of action may involve translocation of the RNA transcript through the hole in its ring structure, its action could depend on the availability of a free 5' terminus. To determine whether Rho's activity is 5'-end-dependent, its ability to bind to and function on a circular derivative of lambda cro mRNA was investigated. The circular derivative was made in vitro by action of RNA ligase on a derivative of lambda cro RNA containing an extra 10-nucleotide sequence near the 5'-end that was complementary to a sequence located near the 3'-end. Rho bound nearly as tightly to the circular derivative RNA as to the standard cro transcript. Rho was also able to readily dissociate a DNA oligonucleotide from its helical complex with the circular RNA in an ATP-dependent reaction. Thus, the action of Rho on a transcript does not depend on the availability of a free 5' terminus.

Adenosine Triphosphate↗
PubMed2000

RNA passes through the hole of the protein hexamer in the complex with the Escherichia coli Rho factor.

Escherichia coli transcription termination factor Rho is a ring-shaped hexameric protein that uses the energy derived from ATP hydrolysis to dissociate RNA transcripts from the ternary elongation complex. To test a current model for the interaction of Rho with RNA, three derivatives of Rho were made containing single cysteine residues and modified with a photo-activable cross-linker. The positions for the cysteines were: 1) in part of the primary RNA-binding site in the N terminus (Cys-82 Rho); 2) in a connecting polypeptide proposed to be on the outside of the hexamer (Cys-153 Rho); and 3) near the proposed secondary RNA-binding site in the ATP-binding domain (Cys-325 Rho). Results from the cross-linking of the modified Rho proteins to a series of lambda cro RNA derivatives showed that Cys-82 Rho formed cross-links with all transcripts containing the Rho utilization (rut) site, that Cys-325 Rho formed cross-links to transcripts that had the rut site and 10 or more residues 3' of the rut site, and that Cys-153 did not form cross-links with any of the transcripts. From a model of the quaternary structure of Rho, which is largely based on homology to the F(1)-ATPase, amino acid 82 is located near the top of the hexamer, and amino acid 325 is located on a solvent-accessible loop in the center of the hexamer. These data are consistent with binding of the rut region of RNA around the crown, with its 3'-segment passing through the center of the Rho hexamer.

Adenosine Triphosphate↗
PubMed2000

The bicyclomycin sensitivities of 38 bicyclomycin-resistant mutants of transcription termination protein rho and the location of their mutations support a structural model of rho based on the F(1) ATPase.

A total of 38 bicyclomycin-resistant mutants of Escherichia coli transcription termination protein Rho have been isolated. The locations of their mutations identify the ATP-binding region as the functional domain inhibited by bicyclomycin. Strains containing the S266C, S266A and L208R Rho mutations are very resistant to bicyclomycin in vivo. In a similar way, the mutant Rho proteins containing these mutations are very resistant to bicyclomycin in vitro. These data suggest that Ser266 and Leu208 might make direct contact with the antibiotic. These two residues are close to each other in the tertiary structure of a model of Rho based on the alpha and beta subunits of the F(1) ATPase, supporting the validity of the model. The strain containing the G337S Rho mutation also has high bicyclomycin resistance, and the proximity of L208, S266 and G337 in the quaternary structure of the Rho model has enabled a candidate bicyclomycin-binding pocket to be delineated. As a whole, the bicyclomycin sensitivities of the mutants are consistent with the locations of their respective mutations in the model of Rho based on the F(1) ATPase, therefore supporting the emerging consensus model of Rho structure.

Adenosine Triphosphate↗
PubMed2000

Determination of the free-energy change for repair of a DNA phosphodiester bond.

The repair of phosphodiester bonds in nicked DNA is catalyzed by DNA ligases. Ligation is coupled to cleavage of a phosphoanhydride bond in a nucleotide cofactor resulting in a thermodynamically favorable process. A free energy value for phosphodiester bond formation was calculated using the reversibility of the T4 DNA ligase reaction. The relative number of DNA nicks to phosphodiester bonds in a circular plasmid DNA, formed during this reaction at fixed concentrations of ATP to AMP and PP(i), was quantified. At 25 degrees C, pH 7, the equilibrium constant (K(eq)) for the ligation reaction is 3.89 x 10(4) m. This value corresponds to a standard free energy (DeltaG degrees ') of -6.3 kcal mol(-1). By subtracting the known energy contribution due to hydrolysis of ATP to AMP and PP(i), DeltaG degrees ' for the hydrolysis of a DNA phosphodiester bond is -5.3 kcal mol(-1).

Adenosine Monophosphate↗
PubMed1999

ATP and other nucleotides stabilize the Rho-mRNA complex.

Transcription termination factor Rho from Escherichia coli is a protein that consists of a single 47 kDa protomeric unit that can form a hexameric structure. To determine whether active hexamers can form on an RNA by assembly of subunits, we measured the dependence of complex formation on the concentration of Rho protein in the presence and absence of various nucleotides and related the binding properties to association states determined from sedimentation properties. The results show that the presence of adenine nucleotides converts RNA binding from a multimeric process to a largely monomeric process and that the change correlates with the stabilization of multimers of Rho by the nucleotides. The experimental evidence also indicates that the hexameric form of Rho is stabilized slightly by binding to a transcript but that the protein on RNA is in equilibrium with nonhexameric forms. These results suggest that a Rho hexamer can form on a transcript by addition of subunits to a partial assembly, which means that the complex can consist of six subunits surrounding an RNA transcript as proposed in recent models for Rho action.

Adenine Nucleotides↗
PubMed1999

Activation of Rho-dependent transcription termination by NusG. Dependence on terminator location and acceleration of RNA release.

There is a kinetic limitation to Rho function at the first intragenic terminator in the lacZ gene (tiZ1) which can be overcome by NusG: Rho can terminate transcription with slowly moving, but not rapidly moving, RNA polymerase unless NusG is also present. Here we report further studies with two other Rho-dependent terminators that are not kinetically limited (tiZ2 and lambda tR1) which show that the requirement for NusG depends on the properties of the terminator and its location in the transcription unit. NusG is also shown to increase the rate of Rho-mediated dissociation of transcription complexes arrested at a specific termination stop point in the tiZ1 region and the rates of dissociation with three different Rho factors and two different terminators correlated with their sensitivity to RNA polymerase elongation kinetics. These results suggest a model of NusG function which involves an alteration in the susceptibility of the transcription complex to Rho action which allows termination to occur within the short kinetic window when RNA polymerase is traversing the termination region.

Bacterial Proteins↗
PubMed1998

Human matrix metalloproteinase-9: activation by limited trypsin treatment and generation of monoclonal antibodies specific for the activated form.

For many studies on matrix metalloproteinases in immunohistochemistry it is important to be able to distinguish between the zymogen and activated forms of the enzymes. Activated human matrix metalloproteinase-9 (MMP-9, gelatinase B) was produced from the proenzyme by limited digestion with trypsin. The products of cleavage were characterised by SDS/PAGE and N-terminal sequencing. Trypsin treatment led to a stepwise removal of the propeptide domain and also caused cleavage within the C-terminal domain. Monoclonal antibodies specific for the activated form of human MMP-9 were raised by using a peptide corresponding to the N-terminus of the activated enzyme as immunogen. The antibodies do not recognise the MMP-9 proenzyme or the active or proenzyme forms of matrix metalloproteinase-2 (MMP-2, gelatinase A) and do not react with unrelated proteins in an unfractionated tissue extract. The antibodies were used to detect, by immunohistochemistry, activated MMP-9 in formalin-fixed, wax-embedded sections from a series of oesophageal cancer cases previously shown to contain MMP-9. All of the tumours contained activated MMP-9 localised to tumour cells and macrophages. As the antibodies are effective in immunohistochemistry on formalin-fixed, wax-embedded sections, they should prove useful for the detection of activated MMP-9 in various disease processes.

Amino Acid Sequence↗
PubMed1998

rut Sites in the nascent transcript mediate Rho-dependent transcription termination in vivo.

The in vitro function of the coliphage lambda tR1 Rho-dependent terminator is governed primarily by a tripartite upstream sequence element designated rut. To determine the contribution of the different components of the rut site to terminator function in the normal context of coupled translation of the nascent cro message, tR1 variants lacking different rut site sequences were tested for terminator function in vivo. Intact rutA and rutB sequences were both necessary for efficient termination. However, deletion of the upstream rutA was far more detrimental than deletion of rutB. The intervening boxB, which encodes a short RNA stem and loop, could be deleted without reducing termination or detectably altering Rho's interaction with the corresponding cro transcript. The relative importance of these sequence elements was also the same in a minimal in vitro termination assay system. Rut sequences are therefore essential for terminator function in vivo and rutA contributes substantially more to tR1 function than does rutB. The relative contribution of these elements can be ascribed to differences in Rho's binding affinity for the encoded transcripts. If other cellular factors also bind the rut element RNA, they do not alter the relative contribution of its two regions to Rho-dependent transcription termination in vivo.

Adenosine Triphosphatases↗
PubMed1998

Combinatorial effects of NusA and NusG on transcription elongation and Rho-dependent termination in Escherichia coli.

The transcription factors NusA and NusG from Escherichia coli are modulators of the RNA polymerase elongation reaction and Rho-dependent transcription termination. NusA decreases the elongation rate and termination efficiency while NusG increases both activities. Both Nus factors are able to physically interact with Rho and with RNA polymerase. Experiments with purified components designed to determine whether these factors act independently or competitively showed that the change in elongation rate was a composite of their individual effects, that the combined effect on termination was dependent on the reaction conditions and that the two factors do not compete for their sites of action for either effect. The two factors were also found not to enhance significantly the slight (20%) inhibition of elongation caused by 200 microM guanosine 3',5'-bisdiphosphate (ppGpp) during transcription in vitro. The results also show that the effects of NusA and NusG on RNA polymerase elongation and Rho function are contrary to the inverse relationship between elongation and termination that is expected for a kinetic coupling of Rho action to RNA polymerase elongation. This property suggests that in addition to their known actions on RNA polymerase that influence the length of pausing, these factors act on some other rate-limiting step of the Rho-dependent termination process.

Bacterial Proteins↗
PubMed1998

Crystal structure of the RNA-binding domain from transcription termination factor rho.

Transcription termination factor rho is an ATP-dependent hexameric helicase found in most eubacterial species. The Escherichia coli rho monomer consists of two domains, an RNA-binding domain (residues 1-130) and an ATPase domain (residues 131-419). The ATPase domain is homologous to the beta subunit of F1-ATPase. Here, we report that the crystal structure of the RNA-binding domain of rho (rho130) at 1.55 A confirms that rho130 contains the oligosaccharide/oligonucleotide-binding (OB) fold, a five stranded beta-barrel. The beta-barrel of rho130 is also surprisingly similar to the N-terminal beta-barrel of F1 ATPase, extending the applicability of F1 ATPase as a structural model for hexameric rho.

Crystallography, X-Ray↗
PubMed1998

The NMR structure of the RNA binding domain of E. coli rho factor suggests possible RNA-protein interactions.

Rho protein is an essential hexameric RNA-DNA helicase that binds nascent mRNA transcripts and terminates transcription in a wide variety of eubacterial species. The NMR solution structure of the RNA binding domain of rho, rho130, is presented. This structure consists of two sub-domains, an N-terminal three-helix bundle and a C-terminal beta-barrel that is structurally similar to the oligosaccharide/oligonucleotide binding (OB) fold. Chemical shift changes of rho130 upon RNA binding and previous mutagenetic analyses of intact rho suggest that residues Asp 60, Phe 62, Phe 64, and Arg 66 are critical for binding and support the hypothesis that ssRNA/ssDNA binding is localized in the beta-barrel sub-domain. On the basis of these studies and the tertiary structure of rho130, we propose that residues Asp 60, Phe 62, Phe 64, Arg 66, Tyr 80, Lys 105, and Arg 109 participate in RNA-protein interactions.

Binding Sites↗
PubMed1997

Function of the novel subdomain in the RNA binding domain of transcription termination factor Rho from Micrococcus luteus.

Transcription termination factor Rho from Micrococcus luteus, a high G + C Gram-positive bacterium, contains an unusual extra sequence within its RNA binding domain that is rich in Arg, Glu, and Asp residues and deficient in hydrophobic residues. To determine the role of this extra sequence, we compared the biochemical properties of a variant lacking nearly all the extra sequence, des(60-300) Rho, to that of wild-type M. luteus Rho. The two forms had very similar properties except that the des(60-300) Rho was unable to terminate transcription with Escherichia coli RNA polymerase at the promoter proximal sites used by the wild-type Rho on a lambda cro DNA template but could cause termination at more distal sites and did cause termination at proximal sites when ITP replaced GTP in the reaction mixture. The RNA binding properties of the two forms of this Rho with normal and inosine-substituted RNAs were found to correlate fully with their termination properties. These results indicate that the arginine-rich extra sequence is directly involved in the selection of the termination site and support the hypothesis that the sequence is present in M. luteus Rho to facilitate its binding to M. luteus transcripts, which are likely to have a high degree of base-paired secondary structure because of their high proportion of G residues.

Adenosine Triphosphatases↗