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Biomedical subjects

M Sprinzl

Publications and source records attributed to M Sprinzl.

At least 127 records · Page 7Linked to original sources
PubMed1983

Interaction of cinnamyl-tRNAPhe with Escherichia coli elongation factor Tu.

The products of nitrous acid mediated-deamination of Phe-tRNAPhe from E. coli were analyzed and their capability to interact with elongation factor Tu from E. coli was investigated. Thin-layer chromatography as well as HPLC analysis revealed the existence of at least two deamination products, 3-phenyl-lactyl-tRNAPhe and cinnamyl-tRNAPhe. It could be shown that the aminoacyl-tRNA analogues were active in the formation of the ternary complex with EF-Tu X GTP, although with a lower efficiency than native Phe-tRNAPhe. For both modified acyl-tRNAs the dissociation constant was determined to be 3 X 10(-5) M.

Chromatography, Gel↗
PubMed1983

Inhibition of ribosomal translocation by peptidyl transfer ribonucleic acid analogues.

The activity of peptidyl-tRNALys-CpCp2'dA was measured in an in vitro poly(A)-dependent polypeptide synthesizing system derived from Escherichia coli. It has already been shown that Lys-tRNALys-CpCp2'dA is active as an acceptor and Ac2-Lys-tRNALys-Cp2'dA can donate its peptidyl residue but that the overall poly(A)-dependent synthesis of polylysine does not take place with Lys-tRNALys-CpCp2'dA [Wagner, T., Cramer, F., & Sprinzl, M. (1982) Biochemistry 21, 1521-1529]. This is due to the efficient inhibition of the EF-G-dependent translocation of the peptidyl-tRNA CpCp2'dA from the ribosomal A to the ribosomal P site. In addition, the EF-G-dependent release of the deacylated tRNALys-CpCp2'dA from the ribosomes is also inhibited. The action of the elongation factor G or some other ribosomal component participating in the translocation process requires the presence of the 2'-hydroxyl group on the terminal adenosine of tRNA. If this hydroxyl group is not present on the tRNA, the ribosomes remain locked in their pretranslocational state.

Deoxyadenosines↗
PubMed1982

On the properties of immobilized elongation factor Tu from Thermus thermophilus HB8.

Elongation factor Tu from Thermus thermophilus was coupled to cyanogen-bromide-activated Sepharose 4B and its properties were investigated. The immobilized elongation factor retained its ligand binding properties. It specifically binds GDP and GTP but does not interact with other nucleotide 5'-phosphates. A conversion of immobilized EF-Tu . GDP to EF-Tu . GTP can be achieved by simple equilibration with GTP. The immobilized EF-Tu . GTP specifically binds aminoacyl-tRNAs and allows a facile purification of aminoacyl-tRNA species from bulk tRNA. It is stable at room temperature for several months and can be repeatedly used for aminoacyl-tRNA isolation.

Binding Sites↗
PubMed1981

Properties of native and nicked elongation factor Tu from Thermus thermophilus HB 8.

Two alternative procedures for the isolation of the elongation factor Tu from Thermus thermophilus were compared and the properties of a specifically nicked EF-Tu . GDP were examined in detail. Although the native elongation factor possessed similar catalytic activities in all reactions investigated as the protein isolated by Arai et al. [Eur. J. Biochem. 92, 509-519 and 521-531 (1978)] it could not be crystallized. The nicked EF-Tu, consisting of two associated fragments with molecular weights of 41000 and 8000 respectively, was active in binding GDP, GTP and in the formation of Phe-tRNAPhe . EF-Tu . GTP ternary complex. However, it did not promote poly(U)-dependent synthesis of polyphenylalanine on Escherichia coli ribosomes. The isolated fragment of a molecular weight of about 41000 did not bind GDP. This activity could be reconstituted with the supplement of the small 8000-Mr fragment. It is demonstrated that, in contrast to the native EF-Tu, the nicked EF-Tu forms high-molecular-weight aggregates. Cleavage of the polypeptide chain of EF-Tu from T. thermophilus stimulates the crystallization of this protein.

Chromatography, Gel↗
PubMed1980

Role of the 5'-terminal phosphate of tRNA for its function during protein biosynthesis elongation cycle.

The 5'-terminal phosphate of tRNAPhe from yeast was removed using tRNAPhe lacking its 3'-terminal adenosine. After regeneration of the C-C-A terminus this tRNA was investigated in following reactions: aminoacylation, spontaneous hydrolysis of the amino acid from aminoacyl-tRNA, aminoacyl-tRNA.EF-Tu.GTP ternary complex formation and poly(U)-dependent synthesis of poly(Phe). The absence of the 5'-terminal phosphate of Phe-tRNAPhe does not influence the rate of hydrolysis of the amino acid or the ability of this rRNA to participate in complex formation with EF-Tu.GTP. The translation of the polyuridylic acid is slightly inhibited whereas the rate and extent of the enzymatic aminoacylation is not affected.

Bacterial Proteins↗
PubMed1980

Compilation of tRNA sequences.

This compilation presents in a small space the tRNA sequences so far published. The numbering of tRNAPhe from yeast is used following the rules proposed by the participants of the Cold Spring Harbor Meeting on tRNA 1978 (1,2;Fig. 1). This numbering allows comparisons with the three dimensional structure of tRNAPhe. The secondary structure of tRNAs is indicated by specific underlining. In the primary structure a nucleoside followed by a nucleoside in brackets or a modification in brackets denotes that both types of nucleosides can occupy this position. Part of a sequence in brackets designates a piece of sequence not unambiguosly analyzed. Rare nucleosides are named according to the IUPACIUB rules (for complicated rare nucleosides and their identification see Table 1); those with lengthy names are given with the prefix x and specified in the footnotes. Footnotes are numbered according to the coordinates of the corresponding nucleoside and are indicated in the sequence by an asterisk. The references are restricted to the citation of the latest publication in those cases where several papers deal with one sequence. For additional information the reader is referred either to the original literature or to other tRNA sequence compilations (3-7). Mutant tRNAs are dealt with in a compilation by J. Celis (8). The compilers would welcome any information by the readers regarding missing material or erroneous presentation. On the basis of this numbering system computer printed compilations of tRNA sequences in a linear form and in cloverleaf form are in preparation.

Bacteria↗
PubMed1980

The effect of chemical modification of the CCA end of yeast tRNAPhe on its biological activity on ribosomes.

Yeast tRNAPhe containing 2-thiocytidine (s2C) at position 75 was alkylated specifically at this residue. The biological activities of alkylated and native tRNAPhe were compared in an Escherichia coli protein-synthesizing system in vitro. The alkylated tRNAPhe proved to be active in all steps involved in the elongation phase but the rate of the peptide transfer reaction was somewhat lower when the alkylated tRNAPhe acted as an acceptor of peptidyl residues as compared to native tRNAPhe. These results raise the possibility for attaching spectroscopic or affinity labels at the s2C-75 residue of tRNAPhe without impairing the activity of the tRNA.

Base Sequence↗
PubMed1980

The complex formation between Escherichia coli aminoacyl-tRNA, elongation factor Tu and GTP. The effect of the side-chain of the amino acid linked to tRNA.

The interaction between Escherichia coli aminoacyl-tRNAs and elongation factor Tu (EF-Tu) x GTP was examined. Ternary complex formation with Phe-tRNAPhe and Lys-tRNALys was compared to that with the respective misaminoacylated Tyr-tRNAPhe and Phe-tRNALys. There was no pronounced difference in the efficiency of aminoacyl-tRNA x EF-Tu x GTP complex formation between Phe-tRNAPhe and Tyr-tRNAPhe. However, Phe-tRNALys was bound preferentially to EF-Tu x GTP as compared to Lys-tRNALys. This was shown by the ability of EF-Tu x GTP to prevent the hydrolysis of the aminoacyl ester linkage of the aminoacyl-tRNA species. Furthermore, gel filtration of ternary complexes revealed that the complex formed with the misaminoacylated tRNALys was also more stable than the one formed with the correctly aminoacylated tRNALys. Both misaminoacylated aminoacyl-tRNA species could participate in the ribosomal peptide elongation reaction. Poly(U)-directed synthesis of poly(Tyr) using Tyr-tRNAPhe occurred to a comparable extent as the synthesis of poly(Phe) with Phe-tRNAPhe. In the translation of poly(A) using native Lys-tRNALys, poly(Lys) reached a lower level than poly(Phe) when Phe-tRNALys was used. It was concluded that the side-chain of the amino acid linked to a tRNA affects the efficiency of the aminoacyl-tRNA x EF-Tu x GTP ternary complex formation.

Amino Acyl-tRNA Synthetases↗
PubMed1979

Proton nuclear magnetic resonance of minor nucleosides in yeast phenylalanine transfer ribonucleic acid. Conformational changes as a consequence of aminoacylation, removal of the Y base, and codon--anticodon interaction.

The assignments of the resonances of the methyl and methylene groups belonging to the residues dihydro-uridine-16 and -17 (C5 and C6), dimethylguanosine-26, N-2-methylguanosine-10, and 7-methylguanosine-46 of yeast tRNAPhe at low temperature are reported. Observing the high-field proton NMR spectral region at different temperatures, the effects of aminoacylation, removal of the Y base, and codon-anticodon interaction on the tertiary structure of yeast tRNAPhe were investigated. The following are the results of this study. (1) The two dihydrouridine residues of tRNAPhe have different environments in aqueous solution: dihydro-uridine-16 is more shielded than dihydrouridine-17. (2) The ribothymidine residue from the fragment (47--76) of yeast tRNAPhe and from a tRNA with a partially disrupted structure exhibits multiple conformations arising from different stacking modes between the ribothymidine-54 and the guanosine-53 residue. (3) Upon aminoacylation the type of guanosine-53 interaction with ribothymidine-54 in the tRNAPhe changes. (4) Removal of the Y base from the anticodon loop of yeast tRNAPhe weakens the thermal stability of the tertiary interactions. (5) The interaction of two complementary anticodons in the absence of proteins and of ribosomes results in stabilization of the tertiary structure. Codon-anticodon interaction dependent rearrangement of the tertiary structure of yeast tRNAPhe was not observed. The spin-lattice relaxation times of the methyl and methylene groups of the minor nucleosides in yeast tRNAPhe demonstrate that the minor nucleosides undergo rotational reorientation (tau c) in the nano-second range. The observed differences in these tau c values indicate a similarity of structure of tRNAPhe in solution and in crystalline form.

Anticodon↗