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PubMed2001

HIV-1 nucleocapsid protein zinc finger structures induce tRNA(Lys,3) structural changes but are not critical for primer/template annealing.

Retroviral reverse transcriptases use host cellular tRNAs as primers to initiate reverse transcription. In the case of human immunodeficiency virus type 1 (HIV-1), the 3' 18 nucleotides of human tRNA(Lys,3) are annealed to a complementary sequence on the RNA genome known as the primer binding site (PBS). The HIV-1 nucleocapsid protein (NC) facilitates this annealing. To understand the structural changes that are induced upon NC binding to the tRNA alone, we employed a chemical probing method using the lanthanide metal terbium. At low concentrations of NC, the strong terbium cleavage observed in the core region of the tRNA is significantly attenuated. Thus, NC binding first results in disruption of the tRNA's metal binding pockets, including those that stabilize the D-TPsiC tertiary interaction. When NC concentrations approach the amount needed for complete primer/template annealing, NC further destabilizes the tRNA acceptor-TPsiC stem minihelix, as evidenced by increased terbium cleavage in this domain. A mutant form of NC (SSHS NC), which lacks the zinc finger structures, is able to anneal tRNA(Lys,3) efficiently to the PBS, and to destabilize the tRNA tertiary core, albeit less effectively than wild-type NC. This mutant form of NC does not affect cleavage significantly in the helical regions, even when bound at high concentrations. These results, as well as experiments conducted in the presence of polyLys, suggest that in the absence of the zinc finger structures, NC acts as a polycation, neutralizing the highly negative phosphodiester backbone. The presence of an effective multivalent cationic peptide is sufficient for efficient tRNA primer annealing to the PBS.

Amino Acid Sequence↗

PubMed1998

Structure/function correlation of spermine-analogue-induced modulation of peptidyltransferase activity.

In a cell-free system derived from Escherichia coli, various analogues of spermine were used to study their effect on the binding of AcPhe-tRNA to poly (U)-programmed ribosomes and on the puromycin reaction carried out at 6 mM Mg2+ (Ac, acetyl). In the absence of factors washable from ribosomes (FWR fraction), mono-acylated or di-acylated analogues of spermine stimulate the binding of AcPhe-tRNA to a lesser degree than spermine, in the order: N1-acetylspermine > N1,N12-diacetylspermine approximately = N1,N12-dipivaloylspermine. Also, the above analogues do not show any sparing effect on Mg2+ requirements for AcPhe-tRNA binding to ribosomes, in contrast to spermine. The presence of FWR fraction during the binding or acetylation of the secondary amines of spermine moderates or abolishes the stimulatory effect. In addition, all analogues tested enhance the stability of the ternary complex AcPhe-tRNA-poly(U)-ribosome and the extent of AcPhe-puromycin synthesis, particularly in the absence of the FWR fraction. At the kinetic phase of AcPhe-puromycin synthesis, the analogues display both stimulatory and inhibitory effects, depending on the absence (partial noncompetitive inhibition) or the presence of the FWR fraction (nonessential activation in concert with partial noncompetitive inhibition). Detailed kinetic analysis shows that the analogues tested can mimic the behaviour of spermine, however, the potency to affect the peptidyltransferase activity depends on their degree of acylation, acyl-substituent size, charge distribution and on their chain flexibility.

Escherichia coli↗

PubMed2000

Effects of anticodon 2'-O-methylations on tRNA codon recognition in an Escherichia coli cell-free translation.

The methylation of 2'-hydroxyl groups is one of the most common posttranscriptional modifications of naturally occurring stable RNA molecules. Some tRNA species have a 2'-O-methyl nucleoside at the first position of the anticodon, and it was suggested that this modification stabilizes the codon-anticodon duplex. However, no tRNA species have been found to have the modification at the second or third position of the anticodon. In the present study, we measured the effects of anticodon 2'-O-methylation on the codon-reading efficiencies of the anticodon variants of the unmodified forms of Escherichia coli tRNA1(Ser), using a cell-free protein synthesis assay. The modification of C in the first position of the anticodon into 2'-O-methylcytidine increased the efficiency of reading the G-ending codon. On the other hand, the modifications of the second and/or third positions were detrimental to the codon-reading activity. Thus, 2'-hydroxyl groups at the second and third positions of the anticodon may have some role in the translation reaction, and this may be the reason why 2'-O-methyl nucleosides are not found in these positions within natural tRNA species.

Amino Acid Sequence↗

PubMed2006

Hierarchical network between the components of the multi-tRNA synthetase complex: implications for complex formation.

The macromolecular tRNA synthetase complex consists of nine different enzymes and three non-enzymatic factors. This complex was recently shown to be a novel signalosome, since many of its components are involved in signaling pathways in addition to their catalytic roles in protein synthesis. The structural organization and dynamic relationships of the components of the complex are not well understood. Here we performed a systematic depletion analysis to determine the effects of structural intimacy and the turnover of the components. The results showed that the stability of some components depended on their neighbors. Lysyl-tRNA synthetase was most independent of other components for its stability whereas it was most required for the stability of other components. Arginyl- and methionyl-tRNA synthetases had the opposite characteristics. Thus, the systematic depletion of the components revealed the functional reason for the complex formation and the assembly pattern of these multi-functional enzymes and their associated factors.

Amino Acyl-tRNA Synthetases↗

PubMed1995

Analysis of the role of the KMSKS loop in the catalytic mechanism of the tyrosyl-tRNA synthetase using multimutant cycles.

A mobile loop in tyrosyl-tRNA synthetase, which corresponds to the KMSKS signature sequence of class I aminoacyl-tRNA synthetases, destabilizes the E.Tyr.ATP complex but stabilizes the following E.[Tyr-ATP]not equal to transition state for the formation of E.Tyr-AMP. Three amino acid residues in the mobile loop, K230, K233, and T234, are known to be primarily responsible for these effects. We now analyze the network of interactions between these three amino acids using multiple mutant free energy cycles. The complete characterization of the coupling energies within the mobile loop allows each of the steps leading to the formation of the transition state complex to be dissected into its energetic components. In particular, it is found that, in the absence of a functional mobile loop, there is synergistic coupling between the tyrosine and ATP substrates (i.e., each enhances the binding affinity of the other) which stabilizes the E.Tyr.ATP intermediate preceding the transition state complex. Thus, the mobile loop disrupts the synergism between the ATP and tyrosine substrates, using the ATP binding energy to stabilize the transition state for the reaction. Whereas the net effect of the mobile loop in the E.Tyr.ATP complex results from several conflicting side chain interactions that tend to offset each other, conflicting interactions in the E.[Tyr-ATP]not equal to transition state complex have been minimized and stabilizing pairwise interactions between the K230, K233, and T234 side chains are optimized. The tight coupling between the side chains of K230, K233, and T234 suggests that the mobile loop adopts a highly constrained conformation during formation of the transition state complex. These results quantitatively demonstrate the importance of side chain interactions in enzyme catalysis and illustrate the use of binding energy to stabilize the transition state of a reaction and the presence of unfavorable interactions to destabilize the ground state.

Adenosine Triphosphate↗

PubMed2001

Mg2+-induced tRNA folding.

Mg(2+)-induced folding of yeast tRNA(Phe) was examined at low ionic strength in steady-state and kinetic experiments. By using fluorescent labels attached to tRNA, four conformational transitions were revealed when the Mg(2+) concentration was gradually increased. The last two transitions were not accompanied by changes in the number of base pairs. The observed transitions were attributed to Mg(2+) binding to four distinct types of sites. The first two types are strong sites with K(diss) of 4 and 16 microM. The sites of the third and fourth types are weak with a K(diss) of 2 and 20 mM. Accordingly, the Mg(2+)-binding sites previously classified as "strong" and "weak" can be further subdivided into two subtypes each. Fluorescent transition I is likely to correspond to Mg(2+) binding to a unique strong site selective for Mg(2+); binding to this site causes only minor A(260) change. The transition at 2 mM Mg(2+) is accompanied by substantial conformational changes revealed by probing with ribonucleases T1 and V1 and likely enhances stacking of the tRNA bases. Fast and slow kinetic phases of tRNA refolding were observed. Time-resolved monitoring of Mg(2+) binding to tRNA suggested that the slow kinetic phase was caused by a misfolded tRNA structure formed in the absence of Mg(2+). Our results suggest that, similarly to large RNAs, Mg(2+)-induced tRNA folding exhibits parallel folding pathways and the existence of kinetically trapped intermediates stabilized by Mg(2+). A multistep scheme for Mg(2+)-induced tRNA folding is discussed.

Binding Sites↗

PubMed1980

Fluorescent tRNA derivatives and ribosome recognition.

The use of fluorescent derivatives of tRNAPhe (yeast) in studies on tRNA conformation and on tRNA-ribosome recognition is described. Evidence is presented which indicates that under physiological conditions with respect to ionic strength and Mg2+ concentration, tRNAPhe exists in at least two conformations. The functional significance of this behavior is discussed on the basis of aminoacylation experiments. The investigation of the ribosome complexes of tRNAPhe labeled in the anticodon and D-loops has provided evidence suggesting that the presence of the codon, although not appreciably altering the apparent association constant, leads to qualitatively different complexes in which the tRNA appears to be rigidly bound to the codon even in the P-tRNA to the ribosome occurs in several steps, which take place only in the presence of the proper codon. One or more of these steps may represent codon-induced conformational changes of the tRNA molecule, which constitute the molecular basis of the highly specific binding of the tRNA to the ribosome.

Amino Acyl-tRNA Synthetases↗

PubMed2005

The interaction of mitochondrial translational initiation factor 2 with the small ribosomal subunit.

Bovine mitochondrial translational initiation factor 2 (IF-2(mt)) is organized into four domains, an N-terminal domain, a central G-domain and two C-terminal domains. These domains correspond to domains III-VI in the six-domain model of Escherichia coli IF-2. Variants in IF-2(mt) were prepared and tested for their abilities to bind the small (28S) subunit of the mitochondrial ribosome. The binding of wild-type IF-2(mt) was strong (K(d) approximately 10-20 nM) and was not affected by fMet-tRNA. Deletion of the N-terminal domain substantially reduced the binding of IF-2(mt) to 28S subunits. However, the addition of fMet-tRNA stimulated the binding of this variant at least 2-fold demonstrating that contacts between fMet-tRNA and IF-2(mt) can stabilize the binding of this factor to 28S subunits. No binding was observed for IF-2(mt) variants lacking the G-domain which probably plays a critical role in organizing the structure of IF-2(mt). IF-2(mt) contains a 37-amino acid insertion region between domains V and VI that is not found in the prokaryotic factors. Mutations in this region caused a significant reduction in the ability of the factor to promote initiation complex formation and to bind 28S subunits.

Amino Acid Sequence↗

PubMed2005

Biochemical characterization of the deafness-associated mitochondrial tRNASer(UCN) A7445G mutation in osteosarcoma cell cybrids.

The deafness-associated A7445G mutation in the precursor of mitochondrial tRNA(Ser(UCN)) has been identified in several pedigrees from different ethnic backgrounds. To determine the role of nuclear background in the biochemical manifestation associated with the A7445G mutation, we performed a biochemical characterization of this mutation using cybrids constructed by transferring mitochondria from lymphoblastoid cell lines derived from a New Zealand family into human osteosarcoma mtDNA-less (rho(0)) cells. Compared with three control cybrids, three cybrids derived from an affected matrilineal relative carrying the homoplasmic A7445G mutation exhibited approximately 38-57% decrease in the steady-state level of tRNA(Ser(UCN)), which is less reduced levels than in lymphoblastoid cells in the previous study. Furthermore, approximately 22% reduction in the level of aminoacylation of tRNA(Ser(UCN)) was observed in the mutant cybrid cells. Interestingly, approximately 60-63% decrease of steady-state level of ND6 gene, which belongs to the same precursor as that of tRNA(Ser(UCN)), in cybrid cell lines carrying the A7445G mutation, is more than that observed in lymphoblastoid cells. These observations strongly point out a mechanistic link between the processing defect of the tRNA(Ser(UCN)) precursor and decreased stability of ND6 mRNA precursor. These results also imply the influence of nuclear background on the biochemical phenotype associated with the A7445G mutation.

Cell Line↗

PubMed2001

The RNA world of plant mitochondria.

Mitochondria are well known as the cellular power factory. Much less is known about these organelles as a genetic system. This is particularly true for mitochondria of plants, which subsist with respect to attention by the scientific community in the shadow of the chloroplasts. Nevertheless the mitochondrial genetic system is essential for the function of mitochondria and thus for the survival of the plant. In plant mitochondria the pathway from the genetic information encoded in the DNA to the functional protein leads through a very diverse RNA world. How the RNA is generated and what kinds of regulation and control mechanisms are operative in transcription are current topics in research. Furthermore, the modes of posttranscriptional alterations and their consequences for RNA stability and thus for gene expression in plant mitochondria are currently objects of intensive investigations. In this article current results obtained in the examination of plant mitochondrial transcription, RNA processing, and RNA stability are illustrated. Recent developments in the characterization of promoter structure and the respective transcription apparatus as well as new aspects of RNA processing steps including mRNA 3' processing and stability, mRNA polyadenylation, RNA editing, and tRNA maturation are presented. We also consider new suggestions concerning the endosymbiont hypothesis and evolution of mitochondria. These novel considerations may yield important clues for the further analysis of the plant mitochondrial genetic system. Conversely, an increasing knowledge about the mechanisms and components of the organellar genetic system might reveal new aspects of the evolutionary history of mitochondria.

Base Sequence↗

PubMed1975

Localization of the structural change induced in tRNA fMET (Escherichia coli) by acidic pH.

We have compared the molecular mechanism of thermal unfolding for native tRNA fMet (Escherichia coli) and the denatured species produced by annealing at pH 4.3. Relaxation kinetic measurements reveal that the transitions assigned to melting of TphiC, anticodon, and acceptor stem helices at neutral pH remain essentially unaltered at pH 4.3, but the transition corresponding to coupled melting of tertiary structure and dihydrouridine helix is greatly affected. The Tm of this region is more than 20 degrees higher at pH 4.3 and it has a larger enthalpy formation than in the native state. The transition dynamics are also considerably changed. In contrast to the native structure, tRNA fMet1 and tRNA fMet3 have similar tertiary structure stabilities at pH 4.3. We conclude that the structural difference between native and acid-denatured forms is localized in the tertiary structure-dihydrouridine helix cooperative interaction region of the molecule.

Buffers↗

PubMed1999

The thumb domain of the P51-subunit is essential for activation of HIV reverse transcriptase.

The biologically relevant and active form of human immunodeficiency virus reverse transcriptase is a heterodimer produced in a two-step dimerization process. Dimerization involves first the rapid association of the two subunits, followed by a slow conformational change yielding a fully active form. In the present study, we demonstrate that the interaction between the thumb domain of p51 and the RNase-H domain of p66 plays a major role in an essential conformational change required for proper folding of the primer/template and the tRNA-binding site, for maturation and for activation of heterodimeric reverse transcriptase. A synthetic peptide derived from the sequence within the thumb domain of p51, which forms the interface with the RNase-H domains of p66, binds heterodimeric reverse transcriptase with an apparent dissociation constant in the nanomolar range and selectively inhibits activation of heterodimeric reverse transcriptase with an inhibition constant of 1.2 microM. A detailed study of the mechanism of inhibition reveals that this peptide does not require dissociation of heterodimeric RT for efficient inhibition and does not affect subunit association, but interferes with the conformational change required for activation of heterodimeric reverse transcriptase, resulting in a decrease in the affinity of reverse transcriptase for the tRNA and an increase in the stability of the primer/template/reverse transcriptase complex. We have previously proposed that the dimeric nature of reverse transcriptase represents an interesting target for the design of antiviral agents. On the basis of this work, we propose that the conformational changes involved in the activation of reverse transcriptase similarly represent an important target for the design of novel antiviral compounds.

Amino Acid Sequence↗

PubMed1993

Metal-binding site in a class I tRNA synthetase localized to a cysteine cluster inserted into nucleotide-binding fold.

The 10 class I aminoacyl-tRNA synthetases share a common N-terminal nucleotide-binding fold. Idiosyncratic polypeptide insertions into this fold introduce residues important for activity, including those that interact with the tRNA acceptor helix. The class I Escherichia coli methionyl-tRNA synthetase (L-methionine:tRNA(Met) ligase, EC 6.1.1.10), a 676-amino acid homodimer, was shown previously by others to contain zinc and to have an activity dependent on its presence. We show here by atomic absorption spectroscopy and zinc titrations the presence of 1 mol of zinc per polypeptide. Replacement of zinc with cobalt yields an active enzyme with a visible absorption spectrum characteristic of tetrahedral coordination to sulfur ligands and an intense metal-to-sulfur charge-transfer band at 340 nm. Mapping of the metal-binding site by zinc blotting of recombinant and proteolytic fragments localized the site to a polypeptide insertion between two strands and a beta-sheet in the N-terminal nucleotide-binding fold that contains the catalytic site. Beginning at Cys-145, this insertion contains a Cys-Xaa2-Cys-Xaa9-Cys-Xaa2-Cys motif. Site-directed substitution of these cysteines with serines yielded proteins that were stable but generally devoid of activity. With this result there is now at least one example of a class I and of a class II E. coli tRNA synthetase with a metal-binding domain important for activity inserted into the catalytic domain.

Amino Acid Sequence↗

PubMed1997

Analyses of frameshifting at UUU-pyrimidine sites.

Others have recently shown that the UUU phenylalanine codon is highly frameshift-prone in the 3'(rightward) direction at pyrimidine 3'contexts. Here, several approaches are used to analyze frameshifting at such sites. The four permutations of the UUU/C (phenylalanine) and CGG/U (arginine) codon pairs were examined because they vary greatly in their expected frameshifting tendencies. Furthermore, these synonymous sites allow direct tests of the idea that codon usage can control frameshifting. Frameshifting was measured for these dicodons embedded within each of two broader contexts: the Escherichia coli prfB (RF2 gene) programmed frameshift site and a 'normal' message site. The principal difference between these contexts is that the programmed frameshift contains a purine-rich sequence upstream of the slippery site that can base pair with the 3'end of 16 S rRNA (the anti-Shine-Dalgarno) to enhance frameshifting. In both contexts frameshift frequencies are highest if the slippery tRNAPhe is capable of stable base pairing in the shifted reading frame. This requirement is less stringent in the RF2 context, as if the Shine-Dalgarno interaction can help stabilize a quasi-stable rephased tRNA:message complex. It was previously shown that frameshifting in RF2 occurs more frequently if the codon 3'to the slippery site is read by a rare tRNA. Consistent with that earlier work, in the RF2 context frameshifting occurs substantially more frequently if the arginine codon is CGG, which is read by a rare tRNA. In contrast, in the 'normal' context frameshifting is only slightly greater at CGG than at CGU. It is suggested that the Shine-Dalgarno-like interaction elevates frameshifting specifically during the pause prior to translation of the second codon, which makes frameshifting exquisitely sensitive to the rate of translation of that codon. In both contexts frameshifting increases in a mutant strain that fails to modify tRNA base A37, which is 3'of the anticodon. Thus, those base modifications may limit frameshifting at UUU codons. Finally, statistical analyses show that UUU Ynn dicodons are extremely rare in E.coli genes that have highly biased codon usage.

Arginine↗

PubMed1977

RNA-ligant interactions. (I) Magnesium binding sites in yeast tRNAPhe.

X-ray crystallagraphic studies studies indicate that there are at least four site-specifically bound hydrated Mg2+ ions, [Mg(H2O)n]2+, in yeast tRNAPhe. The size and the octahedral coordination geometry, rather than the charge, of [Mg(H2O)N]2+ appear to be the primary reasons for the specificity of magnesium ions in site-binding and in the stabilization of the tertiary structure of tRNA.

Binding Sites↗

PubMed1984

A cloning strategy for the bacteriophage T4 vs gene.

The product of the bacteriophage T4 vs gene, the tau peptide, has been shown to thermally stabilize temperature-sensitive valyl-tRNA synthetase (EC 6.1.1.9). To clone the bacteriophage T4 vs gene, recombinant pBR322-T4 DNA molecules were used to transform Escherichia coli CP 790302 (valSts). Transformants that grew at 43.5 degrees C on selective medium exhibited some properties indicative of a phage-modified valyl-tRNA synthetase. The data are consistent with successful cloning of the vs gene and atypical modification of the valyl-tRNA synthetase in CP 790302.

Centrifugation, Density Gradient↗

PubMed1992

The chromosomal integration site for the Streptomyces plasmid SLP1 is a functional tRNA(Tyr) gene essential for cell viability.

The genetic element SLP1 exists in nature as a single DNA segment integrated into the genome of Streptomyces coelicolor. Upon mating with Streptomyces lividans, a closely related species, SLP1 undergoes precise excision from its chromosomal site and is transferred into the recipient where it integrates chromosomally. Previous work has shown that integration and excision involve site-specific recombination between a chromosomal site, attB, and a virtually identical sequence, attP, on SLP1. We demonstrate here by means of gene replacement that a tRNA(Tyr) sequence that overlaps part of the attB site of S. lividans is both biologically functional and essential for cell viability. The requirement for this tRNA gene has been used to stabilize the inheritance of a segrationally unstable plasmid in cells lacking a chromosomal attB site. The evolution of an essential DNA locus as an attachment site for a chromosomally integrating genetic element represents a novel mechanism of biological adaptation.

Base Sequence↗

PubMed1999

Studies of the genomic RNA of leukosis viruses: implications for RNA dimerization.

Retroviral particles contain two positive-strand genomic RNAs linked together by noncovalent bonds that can be dissociated under mild conditions. We studied genomic RNAs of wild-type and mutant avian leukosis viruses (ALVs) in an attempt to (i) better understand the site(s) of RNA dimerization, (ii) examine whether the primer binding site (PBS) and tRNA primer are involved in dimerization, and (iii) determine the structure of genomic RNA in protease-deficient (PR(-)) mutants. We showed that extensively nicked wild-type ALV genomic RNAs melt cooperatively. This implies a complex secondary and/or tertiary structure for these RNAs that extends well beyond the 5' dimerization site. To investigate the role of the PBS-tRNA complex in dimerization, we analyzed genomic RNAs from mutant viruses in which the tRNA(Trp) PBS had been replaced with sequences homologous to the 3' end of six other chicken tRNAs. We found the genomic RNAs of these viruses are dimers that dissociate at the same temperature as wild-type viral RNA, which suggests that the identity of the PBS and the tRNA primer do not affect dimer stability. We studied two ALV PR(-) mutants: one containing a large (>1.9-kb) inversion spanning the 3' end of gag and much of pol, rendering it deficient in PR, reverse transcriptase, and integrase, and another with a point mutation in PR. In both of these mutant viruses, the genomic RNA appears to be either primarily or exclusively monomeric. These data suggest that ALV can package its RNA as monomers that subsequently dimerize.

Animals↗