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C Florentz

Publications and source records attributed to C Florentz.

72 records · Page 4Linked to original sources
PubMed1990

Efficient aminoacylation of a yeast tRNA(Asp) transcript with a 5' extension.

A yeast aspartic acid tRNA with a 5' extension of 14 nucleotides was obtained by in vitro transcription with T7 DNA dependent RNA polymerase. This transcript, called extended tRNA(Asp) transcript, retains its aspartylation capacity with the same Km and only three times reduced kcat values as compared to those measured for canonical tRNA(Asp). This result indicates that the 5' extension of the amino acid acceptor stem of tRNA(Asp) does not interfere with recognition by aspartyl-tRNA synthetase. However, in contrast to the wild-type tRNA(Asp) transcript, the 5' extended molecule presents a reduced capacity to be mischarged by arginyl-tRNA synthetase, suggesting the existence of different structural requirements in aspartyl- and arginyl-tRNA synthetases for tRNA(Asp) recognition.

Arginine-tRNA Ligase↗
PubMed1990

Search of essential parameters for the aminoacylation of viral tRNA-like molecules. Comparison with canonical transfer RNAs.

Comparative structural and functional results on the valine and tyrosine accepting tRNA-like molecules from turnip yellow mosaic virus (TYMV) and brome mosaic virus (BMV), and the corresponding cognate yeast tRNAs are presented. Novel experiments on TYMV RNA include design of variant genes of the tRNA-like domain and their transcription in vitro by T7 RNA polymerase, analysis of their valylation catalyzed by yeast valyl-tRNA synthetase, and structural mapping with dimethyl sulfate and carbodiimide combined with graphical modelling. Particular emphasis is given to conformational effects affecting the valylation capacity of the TYMV tRNA-like molecule (e.g., the effect of the U43----C43 mutation). The contacts of the TYMV and BMV RNAs with valyl- and tyrosyl-tRNA synthetases are compared with the positions in the molecules affecting their aminoacylation capacities. Finally, the involvement of the putative valine and tyrosine anticodons in the tRNA-like valylation and tyrosylation reactions is discussed. While an anticodon-like sequence participates in the valine identity of TYMV RNA, this seems not to be the case for the tyrosine identity of BMV RNA despite the fact that the tyrosine anticodon has been shown to be involved in the tyrosylation of canonical tRNA.

Anticodon↗
PubMed1990

Relaxation of a transfer RNA specificity by removal of modified nucleotides.

The molecular recognition of specific transfer RNAs by the appropriate aminoacyl-tRNA synthetase is an important step in determining the accuracy of translation of the genetic message from nucleic acids into proteins. Recent studies using variant tRNAs with specific sequence modifications have indicated particular regions that determine their identity. Here we consider whether the base modifications commonly found in tRNAs contribute to their identity. Although unmodified tRNA(Asp) is charged with aspartate as efficiently as the modified native tRNA, it is mischarged with arginine with considerably increased efficiency. Our results indicate that post-transcriptional modification of tRNAs introduces structural 'anti-determinants', restricting the efficiency with which the tRNAs are charged with inappropriate amino acids.

Arginine↗
PubMed1990

Stimulatory effect of ammonium sulfate at high concentrations on the aminoacylation of tRNA and tRNA-like molecules.

The influence of various salts on the aminoacylation of tRNA(Val) and the tRNA-like structure from turnip yellow mosaic virus RNA by yeast valyl-tRNA synthetase has been studied. As expected, increasing the concentration of salts inhibits the enzymatic reaction. However, in the presence of high concentration of ammonium sulfate, and only this salt, the inhibitory effect is suppressed. Under such conditions, the aminoacylation becomes comparable to that measured in the absence of salt. It was shown that ammonium sulfate affects both the catalytic rate of the reaction and the affinity between valyl-tRNA synthetase and the RNAs. Because the affinity between the partners in the complex is increased when the concentration of the salt is high, it is suggested that hydrophobic effects are involved in tRNA/synthetase interactions.

Amino Acyl-tRNA Synthetases↗
PubMed1990

Conformation in solution of yeast tRNA(Asp) transcripts deprived of modified nucleotides.

A synthetic gene of yeast aspartic acid tRNA with a promoter for phage T7 RNA polymerase was cloned in Escherichia coli. The in vitro transcribed tRNA(Asp) molecules are deprived of modified nucleotides and retain their aspartylation capacity. The solution conformation of these molecules was mapped with chemical structural probes and compared to that of fully modified molecules. Significant differences in reactivities were observed in Pb2+ cleavage of the RNAs and in modification of the bases with dimethyl sulphate. The most striking result concerns C56, which becomes reactive in unmodified tRNA(Asp), indicating the disruption of the C56-G19 base pair involved in the D- and T-loop interaction. The chemical data indicate that unmodified tRNA(Asp) transcripts possess a relaxed conformation compared to that of the native tRNA. This conclusion is confirmed by thermal melting experiments. Thus it can be proposed that post-transcriptional modifications of nucleotides in tRNA stabilize the biologically active conformations in these molecules.

Base Sequence↗
PubMed1989

Structural analogies between the 3' tRNA-like structure of brome mosaic virus RNA and yeast tRNATyr revealed by protection studies with yeast tyrosyl-tRNA synthetase.

Contacts between the tRNA-like domain in brome mosaic virus RNA and yeast tyrosyl-tRNA synthetase have been determined by footprinting with enzymatic probes. Regions in which the synthetase caused protections indicative of direct interaction coincide with loci identified by mutational studies as being important for efficient tyrosylation [Dreher, T. W. & Hall, T. C. (1988) J. Mol. Biol. 201, 41-55]. Additional extensive contacts were found upstream of the core of the tRNA-like structure. In parallel, the contacts of yeast tRNATyr with its cognate synthetase were determined by the same methodology and comparison of protected nucleotides in the two RNAs has permitted the assignment of structural analogies between domains in the viral tRNA-like structure and tRNATyr. Amino acid acceptor stems are similarly recognized by yeast tyrosyl-tRNA synthetase in the two RNAs, indicating that the pseudoknotted fold in the viral RNA does not perturb the interaction with the synthetase. A further important analogy appears between the anticodon/D arm of the L-conformation of tRNAs and a complex branched arm of the viral tRNA-like structure. However, no apparent anticodon triplet exists in the viral RNA. These results suggest that the major determinants for tyrosylation of yeast tRNATyr lie outside the anticodon stem and loop, possibly in the amino acid acceptor stem.

Acylation↗
PubMed1988

Valylation of tRNA-like transcripts from cloned cDNA of turnip yellow mosaic virus RNA demonstrate that the L-shaped region at the 3' end of the viral RNA is not sufficient for optimal aminoacylation.

Clones containing different lengths of cDNA corresponding to the 3' region of turnip yellow mosaic virus RNA were constructed and transcribed in vitro into the corresponding RNAs. Each transcript contained the L-shaped tRNA domain (N = 82) plus (i) in the case of 3 upstream sequences up to N = 93, 109 and 258; and (ii) in all cases an additional 6 nucleotide-stretch at the 5' end derived from the T7 promoter. The valylation of these molecules, as well as that of a fragment (N = 159) purified from viral RNA, was studied. Although all transcripts could be valylated by wheat germ valyl-tRNA synthetase, the 3 shorter fragments showed incomplete charging and slower rates, due mainly to lower Vmax values. Thus, although the tRNA-like L-shaped structure is the functional core permitting amino-acylation, upstream nucleotides between positions 82 and 159 play an important role in allowing the highest rates and levels of valylation. Structural arguments supporting this view are discussed.

Amino Acyl-tRNA Synthetases↗
PubMed1987

3-D graphics modelling of the tRNA-like 3'-end of turnip yellow mosaic virus RNA: structural and functional implications.

The tRNA-like structure of the aminoacylatable 3'-end of turnip yellow mosaic virus (TYMV) RNA was submitted to 3-D graphics modelling. A model of this structure has been inferred previously from both biochemical results and sequence comparisons which presents a new RNA folding feature, the "pseudoknot". It has been verified that this structure can be constructed without compromising accepted RNA stereochemical rules, namely base stacking and preferential 3'-endo sugar pucker. The model has aided interpretation of previous structural mapping experiments using chemical and enzymatic probes, and new accessibilities of residues could be predicted and tested. Pseudoknots have been considered as potential splice sites because they form antiparallel helical segments in a single RNA molecule. We have examined this possibility with the constructed 3-D model and could verify the hypothesis on a structural basis. The model presents a striking similarity with canonical tRNA and allows a valuable comparison between the protection patterns of yeast tRNA(Val) and tRNA-like viral RNA by cognate yeast valyl-tRNA synthetase against structural probes.

Base Sequence↗
PubMed1986

Contact areas of the turnip yellow mosaic virus tRNA-like structure interacting with yeast valyl-tRNA synthetase.

The tRNA-like structure of turnip yellow mosaic virus is known to be efficiently recognized and aminoacylated by valyl-tRNA synthetase. The present work reports domains in the isolated tRNA-like fragment (159 terminal nucleotides at the 3'-end of the two viral RNAs) in contact with purified yeast valyl-tRNA synthetase. These domains were determined in protection experiments using chemical and enzymatic structural probes. In addition, new data, re-enforcing the validity of the tertiary folding model for the native RNA, are given. In particular, at the level of the amino acid accepting arm it was found that the two phosphate groups flanking the three guanine residues of loop I are inaccessible to ethylnitrosourea. This is in agreement with a higher-order structure of this loop involving "pseudo knotting", as proposed by Rietveld et al. (1982). Valyl-tRNA synthetase efficiently protects the viral RNA against digestion by single-strand-specific S1 nuclease at the level of the anticodon loop. With cobra venom ribonuclease, specific for double-stranded regions of RNA, protection was detected on both sides of the anticodon arm and at the 5'-ends of loop I, a region that is involved in the building up of the acceptor arm. Loop II, which is topologically homologous to the T-loop of canonical tRNA was likewise protected. Weak protection was observed between arms I and II, and at the 3'-side of arm V. This arm, located at the 5'-side of arm IV (homologous to the D-arm of tRNA), does not participate in the pseudo-knotted model of the valine acceptor arm. Ethylnitrosourea was used to determine the phosphates of the tRNA-like structure in close contact with the synthetase. These are grouped in several stretches scattered over the RNA molecule. In agreement with the nuclease digestion results, protected phosphates are located in arms I, II, and III. Additionally, this chemical probe permits detection of other protected phosphates on the 3'-side of arm IV and on both sides of arm V. When displayed in the three-dimensional model of the tRNA-like structure, protected areas are localized on both limbs of the L-shaped RNA. It appears that valyl-tRNA synthetase embraces the entire tRNA-like structure. This is reminiscent of the interaction model of canonical yeast tRNAVal with its cognate synthetase.

Alkylation↗
PubMed1982

The tRNA-like structure of turnip yellow mosaic virus RNA: structural organization of the last 159 nucleotides from the 3' OH terminus.

The secondary structure of the isolated tRNA-like sequence (n=159) present at the 3' OH terminus of turnip yellow mosaic virus RNA has been established from partial nuclease digestion with S1 nuclease and T1, CL(3), and Naja oxiana RNases. The fragment folds into a 6-armed structure with two main domains. The first domain, of loose structure and nearest the 5' OH terminus, is composed of one large arm which extends into the coat protein cistron. The second, more compact domain, is composed of the five other arms and most probably contains the structure recognized by valyl-tRNA synthetase. In this domain three successive arms strikingly resemble the T[unk], anticodon, and D arms found in tRNA. Near the amino-acid accepting terminus, however, there is a new stem and loop region not found in standard tRNA. This secondary structure is compatible with a L-shaped three-dimensional organization in which the corner of the L and the anticodon-containing limb are similar to, and the amino-acid accepting region different from, that in tRNA. Ethylnitrosourea accessibility studies have shown similar tertiary structure features in the T[unk] loop of tRNA and in the homologous region of the viral RNA.

Journal Article↗
PubMed1982

Large-scale purification of the 3'-OH-terminal tRNA-like sequence (n = 159) of turnip-yellow-mosaic-virus RNA.

In order to undertake structural and functional studies on the 3'-terminal part of turnip yellow mosaic virus RNA, a structure which can be specifically aminoacylated by valyl-tRNA synthetase, we have developed large-scale methods for purifying the tRNA-like sequence. Several experimental approaches were tested. One procedure was retained enabling us to purify large quantities of the homogeneous tRNA-like fragment. Starting from 1.5 g turnip yellow mosaic virus, one obtains 400 mg RNA, which is partially digested by T1 ribonuclease and which yields 1-2 mg pure tRNA-like fragment after three chromatographic steps: two filtrations on Ultrogel ACA 54 and one reverse-phase chromatography (RPC 5) in the presence of urea. A method has been worked out allowing preparation of 10 mg of the fragment per month. The purified RNA material appeared homogeneous upon polyacrylamide gel electrophoresis under denaturing conditions. The isolated tRNA-like structure can be valylated to an extent of 100% in the presence of purified yeast valyl-tRNA synthetase with kinetic parameters resembling those of the tRNAVal aminoacylation.

Base Sequence↗
PubMedDate not supplied

Exploring the aminoacylation function of transfer RNA by macromolecular engineering approaches. Involvement of conformational features in the charging process of yeast tRNA(Asp).

This report presents the conceptual and methodological framework that presently underlies the experiments designed to decipher the structural features in tRNA important for its aminoacylation by aminoacyl-tRNA synthetases. It emphasizes the importance of conformational features in tRNA for an optimized aminoacylation. This is illustrated by selected examples on yeast tRNA(Asp). Using the phage T7 transcriptional system, a series of tRNA(Asp) variants were created in which conformational elements were modified. It is shown that aspartyl-tRNA synthetase tolerates conformational variability in tRNA(Asp) at the level of the D-loop and variable region, of the tertiary Levitt base-pair 15-48 which can be inverted and in the T-arm in which residue 49 can be excised. However, changing the anticodon region completely abolishes the aspartylation capacity of the variants. Transplanting the phenylalanine identity elements into a different tRNA(Asp) variant presenting conformational characteristics of tRNA(Phe) converts this molecule into a phenylalanine acceptor but is less efficient than wild-type tRNA(Phe). This engineered tRNA completely loses its aspartylation capacity, showing that some aspartic acid and phenylalanine identity determinants overlap. The fact that chimeric tRNA(Asp) molecules with altered anticodon regions lose their aspartylation capacity demonstrates that this region is part of the aspartic acid identity of tRNA(Asp).

Aspartate-tRNA Ligase↗