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At least 1,099 records · Page 61Linked to original sources
PubMed2005

Plant codon optimized cry genes of Bacillus thuringiensis can be expressed as soluble proteins in Escherichia coli BL21 Codon Plus strain as NusA-Cry protein fusions.

For optimal expression of delta-endotoxins from Bacillus thuringiensis in plants, preferential changes in the codon sequences, and reduction in overall AT content in the nucleotide sequence of the genes is important. Reports suggest that sequences with such modifications cannot be overexpressed in bacteria. We report here that the modified genes can be overexpressed in a strain of Escherichia coli carrying extra tRNA genes for some of the codons occurring at high frequency in plant genes and less preferred in E. coli. We also demonstrate that proteins when expressed as fusion products with NusA protein, are obtained as soluble fraction rather than in inclusion bodies. This allows easy and accurate LC50 analysis on insect pests.

Animals↗
PubMed1999

A distinctive RNA fold: the solution structure of an analogue of the yeast tRNAPhe T Psi C domain.

The structure of an analogue of the yeast tRNAPhe T Psi C stem-loop has been determined by NMR spectroscopy and restrained molecular dynamics. The molecule contained the highly conserved modification ribothymidine at its naturally occurring position. The ribothymidine-modified T Psi C stem-loop is the product of the m5U54-tRNA methyltransferase, but is not a substrate for the m1A58-tRNA methyltransferase. Site-specific substitutions and 15N labels were used to confirm the assignment of NOESY cross-peaks critical in defining the global fold of the molecule. The structure is unusual in that the loop folds far over into the major groove of the curved stem. This conformation is stabilized by both stacking interactions and hydrogen bond formation. Furthermore, this conformation appears to be unique among RNA hairpins of similar size. There is, however, a considerable resemblance to the analogous domain in the crystal structure of the full-length yeast tRNAPhe. We believe, therefore, that the structure we have determined may represent an intermediate in the folding pathway during the maturation of tRNA.

Anticodon↗
PubMed1998

Amber suppression in Escherichia coli by unusual mitochondria-like transfer RNAs.

The "cloverleaf" base-pairing pattern was established as the structural paradigm of active tRNA species some 30 years ago. Nevertheless, this pattern does not accommodate the folding of certain mitochondrial tRNAs. For these recalcitrant tRNAs, we have proposed structures having from 5 to 10 base pairs in the anticodon stem rather than the canonical 6. The absence of these types of tRNAs in cytoplasmic translation systems, however, raises the possibility that they may not be bona fide alternate folding patterns for active tRNA molecules. For this reason, we have designed new tRNA genes based on our model of unusual mitochondrial tRNAs, having 7, 8, 9, and 10 base pairs in the anticodon stem with other modifications to the D-stem and connector regions. We show here that these synthetic genes produce tRNAs that actively suppress amber codons in vivo.

Base Sequence↗
PubMed1995

Improved accumulation and activity of ribozymes expressed from a tRNA-based RNA polymerase III promoter.

RNA polymerase III (pol III) transcripts are abundant in all cells. Therefore, pol III promoters may be ideal for expressing high levels of exogenous RNAs, such as antisense RNAs, decoy RNAs and ribozymes, in many different cell types. We have improved accumulation of recombinant RNAs expressed from a human meti tRNA-derived pol III promoter > 100-fold by modifying the 3' terminus of the transcripts to hybridize to the 5' terminus. This terminal duplex includes the 8 nt leader sequence present in the primary wild-type meti tRNA transcript that is normally removed during processing to the mature tRNA. Expression of an anti-HIV ribozyme was analyzed in cells stably transduced with retroviral vectors encoding pol III transcription units containing this modification. High accumulation of recombinant pol III ribozyme transcripts was observed in all cell lines tested. Due to the enhanced transcript accumulation, ribozyme cleavage activity was readily detectable in total RNA extracted from stably transduced human T cell lines. One pol III transcription unit, termed 'TRZ', was optimized further for ribozyme cleavage activity. The improved pol III transcription units reported here may be useful for expressing a variety of functional and therapeutic RNAs.

Base Sequence↗
PubMed1977

Rabbit liver tRNA1Val:I. Primary structure and unusual codon recognition.

The major valine acceptor tRNA1Val from rabbit liver was purified and its nucleotide sequence determined by in vitro [32P] - labeling with T4 phage induced polynucleotide kinase and finger-printing techniques. Its primary structure was found to be identical with the major valine tRNA from mouse myeloma cells. According to the wobble hypothesis this tRNA, which exclusively has an IAC anticodon, should decode the valine codons GUU, GUC and GUA only. However, this tRNA recognizes all four valine codons with a surprising preference for GUG. It is unknown whether this is due to the lack of A37 modification next to the 3' end of the anticodon IAC. The nature of the inosine-guanosine interaction remains to be clarified.

Animals↗
PubMed1977

Initial rate kinetic analysis of the mechanism of initiation complex formation and the role of initiation factor IF-3.

Initial rate kinetics of the formation of ternary complexes of Escherichia coli 30S ribosomal subunits, poly(uridylic acid), and N-acetylphenylalanyl transfer ribonucleic acid in the presence and in the absence of IF-3 are consistent with the hypothesis that the ternary complex is formed through a random order of addition of polynucleotide and aminoacyl-tRNA to separate and independent binding sites on the 30S ribosomes. The transformation of an intermediate into a stable ternary complex which probably entails a rearrangement of the ribosome structure leading to a codon-anticodon interaction represents the rate-limiting step in the formation of the ternary complex. The rate constant of this transformation, as well as the association constants for the formation of the 30S-poly(U) and 30S-N-AcPhe-tRNA binary complexes, are enhanced by the presence of IF-3 which acts as a kinetic effector on reactions which are intrinsic properties of the 30S ribosome. The IF-3-induced modification of these kinetic parameters of the 30S ribosomal subunit can per se explain the effect of IF-3 on protein synthesis without invoking a specific action at the level of the mRNA-ribosome interaction. This seems to be confirmed by the finding that IF-3 can stimulate several-fold the formation of a ternary complex even if one by-passes the ribosome-template binding step by starting with a covalent 30S-polynucleotide binary complex. Furthermore, the above-mentioned changes induced by IF-3 appear to be compatible with the previously proposed idea that the binding of the factor modifies the conformation of the 30S subunit. The random order of addition of substrates determined for the 30S-N-AcPhe-tRNA-poly(U) model system was found to be valid also for the more physiological 30S initiation complex containing poly(A,U.G) and (fMet-tRNA formed at low Mg2+ concentration in the presence of GTP and all three initiation factors.

Escherichia coli↗
PubMed1999

N-terminal and C-terminal modifications affect folding, release from the ribosomes and stability of in vitro synthesized proteins.

Important aspects of translation are release and folding of the synthesized protein into its three-dimensional structure. Studies from our group indicated that during in vitro protein synthesis a large portion of full-length polypeptides apparently accumulated as peptidyl-tRNA on ribosomes. We have also shown that some proteins though released in biologically active form may be inactivated without being degraded. These experiments were carried out by coupled transcription/translation using an Escherichia coli extract in which eukaryotic or prokaryotic test proteins were synthesized from their coding sequence inserted into specific plasmids. Experiments described here were designed to analyze the effects of N-terminal and C-terminal modifications of the coding sequence on the ribosomal release/termination process and on the stability of the newly synthesized protein. Elimination of the leader sequence in two proteins tested, mitichondrial rhodanese and bacterial beta-lactamase, caused an increase in the percentage of polypeptides released from the ribosomes relative to total synthesis. Conversely, an N-terminal extension such as a histidine-lag impaired the ribosomal release process. Also, a hydrophobic N-terminal modification of the synthesized protein reduced release of newly formed protein from the ribosomes. A C-terminal extension of the coding sequence for rhodanese by one amino acid decreased the percentage released polypeptide and furthermore affected the stability of the in vitro formed protein. We propose that a regulatory mechanism exists by which N-terminal and C-terminal sequences of a newly synthesized protein have feed-back effects on the termination factor-mediated release and on the stability of the native three-dimensional structure.

Cell-Free System↗
PubMed1982

E coli tRNAPhe modified at the 3-(3-amino-3-carboxypropyl) uridine with a photoaffinity label is fully functional for aminoacylation and for ribosomal interaction.

E. coli tRNAPhe was modified at its 3-(3-amino-3-carboxypropyl)uridine residue with the N-hydroxysuccinimide ester of N-(4-azido-2-nitrophenyl) glycine. Exclusive modification of this base was shown by two-dimensional TLC analysis of the T1 oligonucleotide and nucleoside products of nuclease digestion. The fully modified tRNA could be aminoacylated to the same level as control tRNA. The aminoacylated tRNA was as active as control tRNA in non-enzymatic binding to the P site of ribosomes, and in EFTu-dependent binding to the ribosomal A site. The functional activity of this photolabile modified tRNA allows it to be used to probe the A and P binding sites on ribosomes and on the other proteins that interact with tRNA. Crosslinking to the ribosomal P site has been shown.

Affinity Labels↗
PubMed1994

On the binding of isolated yeast tRNA(Phe) anticodon arm to Escherichia coli 30S and 70S ribosomes. Guanosine-42 is important for the binding.

Conditions for the reversible binding of isolated anticodon arm of yeast tRNA(Phe) (15-nucleotide, corresponding to nucleotides N28-N42) to the P site of Escherichia coli 30S and 70S ribosomes have been determined. The affinity constant for the anticodon arm binding to 70S ribosomes is shown to be only 30-fold weaker than that for the binding of total tRNA(Phe). The affinities of yeast tRNA(Phe) and the anticodon arm for 30S subunits and of the anticodon arm for the total 70S ribosomes are shown to be equal. These data imply that the anticodon arm moiety of tRNA(Phe) mainly contributes to the tRNA-70S ribosome interaction, i.e., it contributes for the most part to the total free energy of the deacylated tRNA(Phe) interaction with the P site of the 70S ribosome. By taking into account additional contacts in the 3'-CCA end, other contacts in the region besides the anticodon arm and 3'-CCA end are presumably absent. Within the anticodon arm removal of the 3'-end nucleotide corresponding to guanosine-42 in tRNA(Phe) decreases the association constant of the anticodon arm-ribosome interaction 15-fold. Replacement of this guanosine with other nucleosides as well as modification of the ribose moiety (oxidation and reduction) does not affect the affinity. The replacement data are very likely to indicate that G42 affects the anticodon arm affinity by forming a direct contact with ribosome.

Anticodon↗
PubMed2005

Mycobacterium avium enters a state of metabolic dormancy in response to starvation.

Members of the Mycobacterium avium complex (MAC) exhibit a highly effective and biphasic response to starvation, losing less than 90% viability after 2 years in deionized water. During the first adaptive phase of 4-7 days, the bacilli exhibit a burst of lipid catabolism, alteration of mycolate modifications, loss of catalase and urease activities, and a decline in sensitivity to antibiotics. There is also a decline in the protein level of alanine tRNA synthetase (AlaS), and an increase in ribonuclease E (Rne) levels. During the following persistence phase, the bacilli become metabolically dormant. However, with return of nutrients, the cells rapidly respond with increased activity, as determined by reduction of a tetrazolium dye. The primary reservoir for MAC is natural and municipal water, and the metabolic dormancy may be analogous to that of other aquatic organisms, such as vibrio. The organized metabolic shutdown that environmental mycobacteria utilize to survive starvation may have evolved into the host-specific dormancy mechanisms of Mycobacterium tuberculosis.

Adaptation, Physiological↗
PubMed1981

Nucleotide insertion in the anticodon loop of a glycine transfer RNA causes missense suppression.

We have determined the nucleotide sequences of two unusual UGG-suppressing glycine tRNAs from Escherichia coli and, as a result, have discovered a new mechanism for the generation of missense suppressors. The suppressor tRNAs translate UGG but not UGA. Each arose as a consequence of spontaneous mutational alteration of glyT, the gene for the GGA/G-reading glycine tRNA of E. coli. In each mutant tRNA, the change in primary structure involved the insertion of an adenylate residue on the 3' side of the anticodon and the loss of a modification of the uridylate residue at the 5' end of the anticodon. A "shift" of the effective anticodon by one nucleotide in the 3' direction can account for the new coding specificity of these tRNAs.

Anticodon↗
PubMed1982

The tRNA-like structure at the 3' terminus of turnip yellow mosaic virus RNA. Differences and similarities with canonical tRNA.

The 3' terminus of TYMV RNA, which possesses tRNA-like properties, has been studied. A 3' terminal fragment of 112 nucleotides was obtained by cleavage with RNase H after hybridization of a synthetic oligodeoxynucleotide to the viral RNA. The accessibility of cytidine and adenosine residues was probed with chemical modification. Enzymatic digestion studies were performed with RNase T1, nuclease S1 and the double-strand specific RNase from the venom of the cobra Naja naja oxiana. A model is proposed for the secondary structure of the 3' terminal region of TYMV RNA comprising 86 nucleotides. The main feature of this secondary structure is the absence of a conventional acceptor stem as present in canonical tRNA. However, the terminal 42 nucleotides can be folded in a tertiary structure which bears strong resemblance with the acceptor arm of canonical tRNA. Comparison of this region of TYMV RNA with that of other RNAs from both the tymovirus group and the tobamovirus group gives support to our proposal for such a three-dimensional arrangement. The consequences for the recognition by TYMV RNA of tRNA-specific enzymes is discussed.

Base Sequence↗
PubMed2005

A unique tRNA recognition mechanism of Caenorhabditis elegans mitochondrial EF-Tu2.

Nematode mitochondria expresses two types of extremely truncated tRNAs that are specifically recognized by two distinct elongation factor Tu (EF-Tu) species named EF-Tu1 and EF-Tu2. This is unlike the canonical EF-Tu molecule that participates in the standard protein biosynthesis systems, which basically recognizes all elongator tRNAs. EF-Tu2 specifically recognizes Ser-tRNA(Ser) that lacks a D arm but has a short T arm. Our previous study led us to speculate the lack of the D arm may be essential for the tRNA recognition of EF-Tu2. However, here, we showed that the EF-Tu2 can bind to D arm-bearing Ser-tRNAs, in which the D-T arm interaction was weakened by the mutations. The ethylnitrosourea-modification interference assay showed that EF-Tu2 is unique, in that it interacts with the phosphate groups on the T stem on the side that is opposite to where canonical EF-Tu binds. The hydrolysis protection assay using several EF-Tu2 mutants then strongly suggests that seven C-terminal amino acid residues of EF-Tu2 are essential for its aminoacyl-tRNA-binding activity. Our results indicate that the formation of the nematode mitochondrial (mt) EF-Tu2/GTP/aminoacyl-tRNA ternary complex is probably supported by a unique interaction between the C-terminal extension of EF-Tu2 and the tRNA.

Amino Acid Sequence↗
PubMed1984

Strategy for the mass spectrometric verification and correction of the primary structures of proteins deduced from their DNA sequences.

Fast atom bombardment mass spectrometry has been used to confirm and correct regions from the amino acid sequences of three large proteins, glutaminyl- and glycyl-tRNA synthetase from Escherichia coli and methionyl-tRNA synthetase from yeast, whose primary structures had been deduced from the base sequences of their corresponding genes. The strategy is based on a comparison of the molecular weights of the tryptic peptides predicted from all three reading frames of the gene sequences with those determined mass spectrometrically. The experimental molecular weights either match or differ and can be used to assess the correctness of the base sequences, identify errors that lead to frame shifts, premature stop codons, incorrect amino acids, etc., or identify the presence of posttranslational modifications. This method is very fast and requires little material (5-20 nmol).

Amino Acid Sequence↗
PubMed1994

A mutant cysteinyl-tRNA synthetase affecting timing of chromosomal replication initiation in B. subtilis and conferring resistance to a protein kinase C inhibitor.

A Bacillus subtilis mutant spnA95 was isolated as resistant at 30 degrees C to the protein kinase C (PKC) inhibitor, sphinganine, and temperature sensitive for growth. As deduced by flow cytometry measurements, the mutant has a 35% reduced initiation mass at permissive temperature, resulting in initiation of DNA replication much earlier in the cell cycle than in the wild type. This modification is accompanied by a change in cell size, as determined by phase-contrast microscopy and flow cytometry. Therefore, this strain displays the characteristics of a novel cell clock mutant. spnA is a newly identified gene in B.subtilis and was shown to encode a cysteinyl-tRNA synthetase. At non-permissive temperature, the mutant was defective in the synthesis of P70, a protein with several characteristics of PKC (a cysteine-rich protein). As one possibility, we propose that the altered timing of replication may be due to the reduced synthesis of specific cysteine-rich proteins normally involved in controlling chromosomal replication initiation in B. subtilis.

Amino Acid Sequence↗
PubMed2005

Structure of a putative lipoate protein ligase from Thermoplasma acidophilum and the mechanism of target selection for post-translational modification.

Lipoyl-lysine swinging arms are crucial to the reactions catalysed by the 2-oxo acid dehydrogenase multienzyme complexes. A gene encoding a putative lipoate protein ligase (LplA) of Thermoplasma acidophilum was cloned and expressed in Escherichia coli. The recombinant protein, a monomer of molecular mass 29 kDa, was catalytically inactive. Crystal structures in the absence and presence of bound lipoic acid were solved at 2.1 A resolution. The protein was found to fall into the alpha/beta class and to be structurally homologous to the catalytic domains of class II aminoacyl-tRNA synthases and biotin protein ligase, BirA. Lipoic acid in LplA was bound in the same position as biotin in BirA. The structure of the T.acidophilum LplA and limited proteolysis of E.coli LplA together highlighted some key features of the post-translational modification. A loop comprising residues 71-79 in the T.acidophilum ligase is proposed as interacting with the dithiolane ring of lipoic acid and discriminating against the entry of biotin. A second loop comprising residues 179-193 was disordered in the T.acidophilum structure; tryptic cleavage of the corresponding loop in the E.coli LplA under non-denaturing conditions rendered the enzyme catalytically inactive, emphasizing its importance. The putative LplA of T.acidophilum lacks a C-terminal domain found in its counterparts in E.coli (Gram-negative) or Streptococcus pneumoniae (Gram-positive). A gene encoding a protein that appears to have structural homology to the additional domain in the E.coli and S.pneumoniae enzymes was detected alongside the structural gene encoding the putative LplA in the T.acidophilum genome. It is likely that this protein is required to confer activity on the LplA as currently purified, one protein perhaps catalysing the formation of the obligatory lipoyl-AMP intermediate, and the other transferring the lipoyl group from it to the specific lysine residue in the target protein.

Amino Acid Sequence↗
PubMed1993

Phosphorothioates in pre-tRNAs can change the specificities of RNAses P or reduce the cleavage efficiencies.

Several phosphorothioate-modified E coli and yeast pre-tRNAs were synthesized. If this modification included the phosphodiester at the RNase P cleavage site, two different effects were observed. With some pre-tRNAs the RNase P cleavage efficiency was severely reduced, whereas with other pre-tRNAs a new reaction type for RNase P was observed. Unlike the previously studied base or ribose modifications, phosphorothioates resulted in aberrant cleavages at unmodified phosphodiesters. These new sites could be located in the 5'-flank or in the acceptor stem of the tRNA domain. Modified mutants of E coli pre-tRNA(Tyr) with different base pairs at the RNase P cleavage site were cleaved with reduced efficiencies, but no aberrant products were observed.

Base Composition↗