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At least 271 records · Page 15Linked to original sources
PubMed2000

Mutilation of RNA phage Qbeta virus-like particles: from icosahedrons to rods.

Icosahedral virus-like particles (VLPs) of RNA phage Qbeta are stabilized by four disulfide bonds of cysteine residues 74 and 80 within the loop between beta-strands F and G (FG loop) of the monomeric subunits, which determine the five-fold and quasi-six-fold symmetry contacts of the VLPs. In order to reduce the stability of Qbeta VLPs, we mutationally converted the amino acid stretch 76-ANGSCD-81 within the FG loop into the 76-VGGVEL-81 sequence. It led to production in Escherichia coli cells of aberrant rod-like Qbeta VLPs, along with normal icosahedral capsids. The length of the rod-like particles exceeded 4-30 times the diameter of icosahedral Qbeta VLPs.

Allolevivirus↗
PubMed2025

Genome sequencing reveals the impact of pseudoexons in rare genetic disease.

PURPOSE: Advancements in sequencing technologies have significantly improved clinical genetic testing; yet, the diagnostic yield remains around 30% to 40%. Emerging technologies are now being deployed to address the remaining diagnostic gap. METHODS: We tested whether short-read genome sequencing could increase the diagnostic yield in individuals enrolled into the UCI-GREGoR research study, who had suspected Mendelian conditions and prior inconclusive testing. Two other collaborative research cohorts, focused on aortopathy and dilated cardiomyopathy, consisted of individuals who were undiagnosed but had not undergone harmonized prior testing. RESULTS: We sequenced 353 families (754 participants) and found a molecular diagnosis in 54 (15.3%) of them. Of these diagnoses, 55.5% were previously missed because the causative variants were in regions not originally interrogated. In 5 cases, they were deep intronic variants, all of which led to abnormal splicing and pseudoexons, as directly shown by RNA sequencing. All 5 of these variants had inconclusive spliceAI scores. In 26% of newly diagnosed cases, the causal variant could have been detected by exome sequencing reanalysis. CONCLUSION: Genome sequencing can overcome limitations of clinical genetic testing, such as the inability to call intronic variants. Our findings highlight pseudoexons as a common mechanism via which deep intronic variants cause Mendelian disease.

Humans↗
PubMed2024

Model-directed generation of artificial CRISPR-Cas13a guide RNA sequences improves nucleic acid detection.

CRISPR guide RNA sequences deriving exactly from natural sequences may not perform optimally in every application. Here we implement and evaluate algorithms for designing maximally fit, artificial CRISPR-Cas13a guides with multiple mismatches to natural sequences that are tailored for diagnostic applications. These guides offer more sensitive detection of diverse pathogens and discrimination of pathogen variants compared with guides derived directly from natural sequences and illuminate design principles that broaden Cas13a targeting.

CRISPR-Cas Systems↗
PubMed2001

Comparison of polymerase subunits from double-stranded RNA bacteriophages.

The family Cystoviridae comprises several bacteriophages with double-stranded RNA (dsRNA) genomes. We have previously purified the catalytic polymerase subunit (Pol) of one of the Cystoviridae members, bacteriophage phi6, and shown that the protein can catalyze RNA synthesis in vitro. In this reaction, both bacteriophage-specific and heterologous RNAs can serve as templates, but those containing 3' termini from the phi6 minus strands are favored. This provides a molecular basis for the observation that only plus strands, not minus strands, are transcribed from phi6 dsRNA segments in vivo. To test whether such a regulatory mechanism is also found in other dsRNA viruses, we purified recombinant Pol subunits from the phi6-related bacteriophages phi8 and phi13 and assayed their polymerase activities in vitro. The enzymes catalyze template-dependent RNA synthesis using both single-stranded-RNA (ssRNA) and dsRNA templates. However, they differ from each other as well as from phi6 Pol in certain biochemical properties. Notably, each polymerase demonstrates a distinct preference for ssRNAs bearing short 3'-terminal sequences from the virus-specific minus strands. This suggests that, in addition to other factors, RNA transcription in Cystoviridae is controlled by the template specificity of the polymerase subunit.

Amino Acid Sequence↗
PubMed2005

Complete nucleotide sequence of Radish mosaic virus RNA polymerase gene and phylogenetic relationships in the genus Comovirus.

The 3'-terminal part of RNA1 genome segment of Radish mosaic virus (RaMV) including complete RNA polymerase gene was sequenced. The 207 amino acids long polymerase is matured from a polyprotein precursor by cleavage at putative Q/H site by viral protease. The alignment of available amino acid sequences of RNA polymerase genes of comoviruses revealed a closest (55%) identity of RaMV to Red clover mottle virus (RCMV).

Amino Acid Sequence↗
PubMed1977

Characterization of double-stranded ribonucleic acid sequences present in the initial transcription products of rat liver chromatin.

At low ionic strength and with a low exogenous RNA polymerase/DNA ratio, rat liver chromatin directs the synthesis in vitro of RNA sequences rich in double-stranded segments. All the transcripts contain at least one double-stranded sequence. Most of the double-stranded segments are formed by intramolecular base-pairing of inverted complementary sequences separated by a single-stranded loop. They are heterogeneous in size, 35-45% of them being more than 80 nucleotides long. They contain 61-64% G+C, whether synthesized by rat liver RNA polymerase (form B) or Escherichia coli RNA polymerase. The largest double-stranded sequences are found in the largest transcripts, and are the most thermostable. The fidelity of base-matching is better in double-stranded transcripts synthesized on rat liver chromatin by homologous polymerase than in those synthesized on it by a bacterial polymerase, or in those synthesized by either of the two polymerases on pure DNA.

Animals↗
PubMed1994

Continuous in vitro evolution of bacteriophage RNA polymerase promoters.

Rapid in vitro evolution of bacteriophage T7, T3, and SP6 RNA polymerase promoters was achieved by a method that allows continuous enrichment of DNAs that contain functional promoter elements. This method exploits the ability of a special class of nucleic acid molecules to replicate continuously in the presence of both a reverse transcriptase and a DNA-dependent RNA polymerase. Replication involves the synthesis of both RNA and cDNA intermediates. The cDNA strand contains an embedded promoter sequence, which becomes converted to a functional double-stranded promoter element, leading to the production of RNA transcripts. Synthetic cDNAs, including those that contain randomized promoter sequences, can be used to initiate the amplification cycle. However, only those cDNAs that contain functional promoter sequences are able to produce RNA transcripts. Furthermore, each RNA transcript encodes the RNA polymerase promoter sequence that was responsible for initiation of its own transcription. Thus, the population of amplifying molecules quickly becomes enriched for those templates that encode functional promoters. Optimal promoter sequences for phage T7, T3, and SP6 RNA polymerase were identified after a 2-h amplification reaction, initiated in each case with a pool of synthetic cDNAs encoding greater than 10(10) promoter sequence variants.

Bacteriophage T3↗
PubMed1994

The functional roles of disulfide bonds in the beta-subunit of (Na,K)ATPase as studied by site-directed mutagenesis.

The beta-subunit of Torpedo californica (Na,K)ATPase contains seven cysteine residues; one (Cys46) is in the single transmembrane segment and the other six (Cys127, Cys150, Cys160, Cys176, Cys215 and Cys278) are in the extracellular domain and form three highly conserved disulfide bonds. A beta-subunit mutant with replacement of Cys46 by Ser could assemble with the alpha-subunit, and the resulting alpha beta-complex was catalytically active. Mutants in which either the N-terminal side or both Cys residues of the Cys127-Cys150 bond were replaced by Ser could also tightly assemble with the alpha-subunit, but the resulting alpha beta-complex was catalytically inactive. On the other hand, disruption of either the Cys160-Cys176 or Cys215-Cys278 bond by substituting the N-terminal side only or both Cys residues with Ser led to a beta-subunit that could not assemble with the alpha-subunit. We conclude that the structure of the beta-subunit around the Cys160-Cys176 and Cys215-Cys278 loops is indispensable for assembly with the alpha-subunit, whereas the Cys127-Cys150 loop is not essential for assembly but is required for enzyme activity.

Amino Acid Sequence↗
PubMed2001

A general approach for the generation of orthogonal tRNAs.

BACKGROUND: The addition of new amino acids to the genetic code of Escherichia coli requires an orthogonal suppressor tRNA that is uniquely acylated with a desired unnatural amino acid by an orthogonal aminoacyl-tRNA synthetase. A tRNA(Tyr)(CUA)-tyrosyl-tRNA synthetase pair imported from Methanococcus jannaschii can be used to generate such a pair. In vivo selections have been developed for selecting mutant suppressor tRNAs with enhanced orthogonality, which can be used to site-specifically incorporate unnatural amino acids into proteins in E. coli. RESULTS: A library of amber suppressor tRNAs derived from M. jannaschii tRNA(Tyr) was generated. tRNA(Tyr)(CUA)s that are substrates for endogenous E. coli aminoacyl-tRNA synthetases were deleted from the pool by a negative selection based on suppression of amber nonsense mutations in the barnase gene. The remaining tRNA(Tyr)(CUA)s were then selected for their ability to suppress amber nonsense codons in the beta-lactamase gene in the presence of the cognate M. jannaschii tyrosyl-tRNA synthetase (TyrRS). Four mutant suppressor tRNAs were selected that are poorer substrates for E. coli synthetases than M. jannaschii tRNA(Tyr)(CUA), but still can be charged efficiently by M. jannaschii TyrRS. CONCLUSIONS: The mutant suppressor tRNA(Tyr)(CUA) together with the M. jannaschii TyrRS is an excellent orthogonal tRNA-synthetase pair for the in vivo incorporation of unnatural amino acids into proteins. This general approach may be expanded to generate additional orthogonal tRNA-synthetase pairs as well as probe the interactions between tRNAs and their cognate synthetases.

Amino Acyl-tRNA Synthetases↗
PubMed1998

The CCA-adding enzyme has a single active site.

The CCA-adding enzyme (tRNA nucleotidyltransferase) synthesizes and repairs the 3'-terminal CCA sequence of tRNA. The eubacterial, eukaryotic, and archaeal CCA-adding enzymes all share a single active-site signature motif, which identifies these enzymes as belonging to the nucleotidyltransferase superfamily. Here we show that mutations at Asp-53 or Asp-55 of the Sulfolobus shibatae signature sequence abolish addition of both C and A, demonstrating that a single active site is responsible for addition of both nucleotides. Mutations at Asp-106 (and to a lesser extent, at Glu-173 and Asp-215) selectively impaired addition of A, but not C. We have previously demonstrated that the tRNA acceptor stem remains fixed on the surface of the CCA-adding enzyme during C and A addition (Shi, P.-Y., Maizels, N., and Weiner, A. M. (1998) EMBO J. 17, 3197-3206). Taken together with this new evidence that there is a single active site for catalysis, our data suggest that specificity of nucleotide addition is determined by a process of collaborative templating: as the single active site catalyzes addition of each nucleotide, the growing 3'-end of the tRNA would progressively refold to create a binding pocket for addition of the next nucleotide.

Adenosine Triphosphate↗
PubMed2002

Evidence for a stabilizer element in the untranslated regions of Drosophila glutathione S-transferase D1 mRNA.

The neighboring genes gstD1 and gstD21 share 70% sequence identity. gstD1 encodes a 1,1,1-trichloro-2,2-bis-(P-chlorophenyl)ethane dehydrochlorinase; gstD21, a ligandin. Both of their mRNAs are inducible by pentobarbital but otherwise behave very differently. Intact gstD21 mRNA is intrinsically labile, but becomes stabilized when separated from its native untranslated region (UTR). In contrast, whereas gstD1 mRNA is very stable in its entirety, without its native UTRs it becomes even more labile than that of gstD21. Decay patterns from four chimeric D1-D21 mRNAs, designed to reveal the individual importance of each molecular region to stability, strongly indicate the presence of destabilizing elements in the coding region of gstD1 mRNA. Thus, the UTRs of this molecule must contain a dominant stabilizer element that overrides the destabilizing influence of the coding region and confers overall stability to the entire molecule. The suspected presence of such a stabilizer element in gstD1 mRNA extends a concept from mRNA metabolism in yeast and cultured mammalian cells to include a multicellular organism, Drosophila melanogaster. The complementary presence of destabilizing and stabilizer elements on the same mRNA reveals a regulatory mechanism by which an abundant mRNA can be further induced by a chemical stimulus, or otherwise be returned to normal levels during recovery.

Amino Acid Sequence↗
PubMed2006

Insights to substrate binding and processing by West Nile Virus NS3 protease through combined modeling, protease mutagenesis, and kinetic studies.

West Nile Virus is becoming a widespread pathogen, infecting people on at least four continents with no effective treatment for these infections or many of their associated pathologies. A key enzyme that is essential for viral replication is the viral protease NS2B-NS3, which is highly conserved among all flaviviruses. Using a combination of molecular fitting of substrates to the active site of the crystal structure of NS3, site-directed enzyme and cofactor mutagenesis, and kinetic studies on proteolytic processing of panels of short peptide substrates, we have identified important enzyme-substrate interactions that define substrate specificity for NS3 protease. In addition to better understanding the involvement of S2, S3, and S4 enzyme residues in substrate binding, a residue within cofactor NS2B has been found to strongly influence the preference of flavivirus proteases for lysine or arginine at P2 in substrates. Optimization of tetrapeptide substrates for enhanced protease affinity and processing efficiency has also provided important clues for developing inhibitors of West Nile Virus infection.

Amino Acid Sequence↗
PubMed1992

Analysis of the catalytic specificity of cytochrome P450 enzymes through site-directed mutagenesis.

The way in which structural diversity encodes the capacity of individual P450 enzymes to metabolize multiple, structurally distinct substrates remains largely unknown. The tools of molecular biology provide a means of identifying amino acid residues among closely related P450s that are determinants of their distinct catalytic properties. Work in our laboratory has identified two substrate specificity-determining segments of the amino acid sequences of subfamily 2C P450s. A pattern has emerged from this work, and that of others, which suggests a model for the structural basis of P450 catalytic diversity.

Amino Acid Sequence↗
PubMed1995

The positively acting amdA gene of Aspergillus nidulans encodes a protein with two C2H2 zinc-finger motifs.

Semi-dominant mutations in the amdA gene lead to elevated expression of the gene encoding acetamidase, amdS. These mutations also cause constitutive expression of the acetate-inducible gene, aciA. In the amdS 5' regulatory region, two cis-acting mutations, amdl66 and amdl666, have been isolated which specifically affect amdA activation of amdS. These mutations are a duplication and a triplication of an 18 bp GA-rich sequence, thought to define the amdA site of action within the amdS promoter region. Similar GA-rich sequences have also been found in the 5' region of aciA. This paper describes the cloning and initial functional characterization of the amdA gene and two of its mutant alleles. The wild-type amdA gene has been cloned by a chromosome walk from genes gatA and alcC on linkage group VII and localized by complementation of an amdA loss-of-function mutation. Transcriptional analysis reveals that the gene is expressed constitutively at low levels under growth conditions which affect expression of amdS and aciA. The gene is predicted to encode an 880-amino-acid protein which contains two C2H2 zinc fingers, a nuclear localization sequence and two transcriptional activation domains. The amdA7 semi-dominant gain-of-function mutation results in a glycine to aspartate substitution which would increase the acidity of one of these regions. Analysis of in vitro generated mutations in the 5' region of amdS using an amdS::lacZ reporter has been used to localize the site of action of AmdA. The C2H2 zinc-finger motifs identified in the protein are similar to those found in the carbon catabolite repressor protein, CreA, which also regulates amdS and recognizes sequences which overlap with the proposed site of action for AmdA.

Acetates↗