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Biomedical subjects

F Lustig

Publications and source records attributed to F Lustig.

12 recordsLinked to original sources
PubMed1995

Glycine codon discrimination and the nucleotide in position 32 of the anticodon loop.

Using an in vitro protein-synthesizing system that allowed us to monitor separately the reading of each glycine codon, we have previously shown, that in constructs based on glycine tRNA1 from Escherichia coli the nature of the nucleotide in position 32 determines the ability of the anticodon UCC to discriminate between the glycine codons. Thus, with a U in position 32 the anticodon UCC discriminated according to the wobble rules, but with a C in this position it had lost its ability to discriminate. In the present paper we show that the same is true also for constructs based on mycoplasma glycine tRNA. When C32 in the wild type was changed to U32, the anticodon UCC discriminated between the glycine codons, while in wild type mycoplasma glycine tRNA it did not. Furthermore, when U32 was changed to C32 in glycine tRNA1(CCC), the anticodon CCC loses its ability to discriminate. We therefore conclude that the nature of the nucleotide in position 32 determines the discriminatory ability of both anticodons UCC and CCC in the glycine tRNA1 structural background, and that the same is true for the anticodon UCC in the mycoplasma glycine tRNA background.

Anticodon↗
PubMed1995

Binding of platelet-derived growth factor and low density lipoproteins to glycosaminoglycan species produced by human arterial smooth muscle cells.

The platelet-derived growth factor (PDGF) binds via a defined amino acid sequence to heparin (Fager et al., 1992, In Vitro Cell. Dev. Biol., 28A:176-180) and the protein moiety of low density lipoproteins (LDL; apo B-100) via a similar sequence to chondroitin sulfate (Camejo et al., 1988, Arteriosclerosis Thromb., 8:368-377). In this study, synthetic oligopeptides were used to explore the capacity of smooth muscle cell-derived glycosaminoglycans to bind to the critical sequences of PDGF and apo B-100. In vitro, proliferating human arterial smooth muscle cells synthesized twice as much proteoglycans as did quiescent cells. The dominating glycosaminoglycan side chains were chondroitin and heparan sulfates in secreted and cell-associated proteoglycans, respectively. The chondroitin sulfate-rich proteoglycans had a higher molecular size and were to a larger extent secreted into the culture medium than the heparan and dermatan sulfate-rich proteoglycans. Heparan, dermatan, and chondroitin sulfates bound to the PDGF-derived oligopeptide with affinities similar to those of heparin. However, while heparan and dermatan sulfates both inhibited DNA synthesis in human arterial smooth muscle cells, chondroitin sulfate had no such inhibitory effect. Like the PDGF-derived oligopeptide, the apo B-100-derived oligopeptide bound to these glycosaminoglycans. At the same time, both oligopeptides displaced bound LDL from chondroitin sulfate in vitro and released the block on DNA synthesis in smooth muscle cells that heparin induced in culture. Thus, chondroitin, dermatan, and heparan sulfates produced by arterial smooth muscle cells may bind LDL and PDGF competitively in atherogenesis but only heparan and dermatan sulfates inhibit cellular DNA synthesis. LDL and PDGF deposition may occur by binding to similar binding sites on glycosaminoglycans derived from smooth muscle cells within atherosclerotic lesions.

Apolipoprotein B-100↗
PubMed1990

Codon reading properties of an unmodified transfer RNA.

We have previously shown that the Mycoplasma mycoides glycine tRNA (anticodon UCC) effectively reads the codons GGU and GGC in violation of the classic codon reading rules. We have attempted to elucidate what structural elements in this tRNA molecule confer this translational property and in the course of this investigation T7 RNA polymerase transcription of the corresponding gene was used to produce a tRNA devoid of modified nucleosides. Using an in vitro translation system the ability of this tRNA to read the 4 glycine codons (GGU, GGC, and GGG) was tested and it was shown to be as efficient as its normal, fully modified counterpart in the reading of all four codons. This result demonstrates that a tRNA devoid of modified nucleosides is able to efficiently sustain protein synthesis in vitro and, furthermore, that the normal modification pattern of the Mycoplasma glycine tRNA is not essential for the ability of this tRNA to read the glycine codons GGU and GGC effectively.

Base Sequence↗
PubMed1989

Codon discrimination and anticodon structural context.

Site-directed mutagenesis has been used to change the nucleotide C in the wobble position of tRNA(1Gly) (CCC) to U. The mutated tRNA was tested for its ability to read glycine codons in an in vitro protein-synthesizing system programmed with the phage message MS2-RNA that had been modified by site-directed mutagenesis so as to make it possible to monitor conveniently the reading of all four glycine codons. The results showed that while the efficiency of tRNA(1Gly) (UCC) was comparable to that of mycoplasma tRNA(Gly) (UCC) in the reading of the codon GGA, the mycoplasma tRNA(Gly) was far more efficient than the tRNA(1Gly) (UCC) in the reading of the codons GGU and GGC. Thus, the anticodon UCC, when present in the structural context of the tRNA(1Gly) molecule, behaved as predicted by the wobble rules while in the structural context of the mycoplasma tRNA(Gly) it read without discrimination between the nucleotides in the third codon position, in violation of the wobble restrictions. The result with the codon GGG showed that the anticodon UCC, when present in tRNA(1Gly), was considerably less efficient in reading this codon than it was in the structural context of the mycoplasma tRNA(Gly). It would therefore seem that the anticodon UCC, when present in a certain tRNA, can be an efficient wobbler, while in the molecular environment of another tRNA it is markedly restricted in its ability to wobble.

Amino Acid Sequence↗
PubMed1988

Properties of a transfer RNA lacking modified nucleosides.

A transfer RNA complete devoid of modified nucleosides was synthesized by in vitro transcription, and some of its properties in aminoacylation and protein synthesis in vitro were studied. For this purpose, a plasmid was constructed which contained a glycine tRNA gene from Mycoplasma mycoides under the promoter of the T7 RNA polymerase, as well as a BstNI restriction site at the 3'-end of the tRNA gene. Cleavage of plasmid DNA with BstNI followed by T7 RNA polymerase transcription in vitro yielded an RNA which was processed with M1 RNA, the catalytic subunit of ribonuclease P, to give a tRNA of mature length. The tRNA synthesized in this manner can be esterified with glycine in vitro, and the rate of aminoacylation is the same as when using the corresponding fully modified glycine tRNA from M. mycoides. Furthermore, in protein synthesis in vitro, the tRNA lacking modified nucleosides was essentially as efficient as the corresponding normal glycine tRNA. However, the Escherichia coli extract used in our protein-synthesizing system introduced one modification, pseudouridine, into the in vitro-synthesized tRNA, and it cannot be excluded that this modification has an essential role in protein synthesis.

Adenosine↗
PubMed1987

Apparent lack of discrimination in the reading of certain codons in Mycoplasma mycoides.

We report a cluster of four tRNA genes from Mycoplasma mycoides as well as the sequence of the alanine, proline, and valine tRNAs and the serine tRNA reading the UCN codons (where N stands for G, A, C, or U). This brings the total number of tRNA genes that we have so far characterized in this organism to 14, 6 of which code for tRNAs that read the codons of family boxes. In each of these latter cases, we found only one gene per family box, and the gene sequence contains a thymidine in the position corresponding to the wobble nucleotide, with the exception of the arginine tRNA gene that has an adenosine in this position. Furthermore, all of the tRNA structures reported here have an unsubstituted uridine in the wobble position. These findings are similar to those reported for mitochondria, especially yeast mitochondria, that contain an arginine tRNA with the anticodon ACG. However, the resemblance is not complete since we have demonstrated the presence of two isoacceptor tRNAs for threonine having uridine and adenosine, respectively, in the wobble position. It is suggested that in the M. mycoides at least some of the family codon boxes are read by only one tRNA each, using an unconventional method without discrimination between the nucleotides in the third codon position.

Base Sequence↗
PubMed1985

Cloning and nucleotide sequence analysis of transfer RNA genes from Mycoplasma mycoides.

As part of an investigation of the tRNA genes of Mycoplasma mycoides, two HindIII fragments of mycoplasma DNA comprising 0.4 and 2.5 kilobases (kb), respectively, were cloned in pBR322 and their nucleotide sequences determined. Only one tRNA gene was found in the 0.4 kb fragment, the gene for tRNAArg with the anticodon TCT, while the 2.5 kb fragment contained nine different tRNA genes arranged in a cluster which presumably constitutes a transcriptional unit. The clustered tRNA genes, with their respective anticodons, were as follows: Arg (ACG), Pro (TGG), Ala (TGC), Met (CAT), Ile (CAT), Ser (TGA), fMet (CAT), Asp (GTC), and Phe (GAA).

Base Sequence↗
PubMed1981

Codon reading and translational error. Reading of the glutamine and lysine codons during protein synthesis in vitro.

The reading of glutamine and lysine codons during protein synthesis in vitro has been investigated using an MS2-RNA-programed system derived from Escherichia coli. Under conditions when either glutaminyl-tRNA1Gln (s2UUG) or glutaminyl-tRNA2Gln (CUG) was the only source of glutamine for protein synthesis both tRNAs were able to read the glutamine codons CAA and CAG as indicated by the incorporation of labeled glutamine into the pertinent coat protein tryptic peptides. On the other hand, when the two glutamine tRNAs competed for the codon CAA the reading efficiency of the anticodon s2UUG, which reads the codon according to the wobble rules, was almost 40 times higher than that of the competing anticodon CUG, which reads the codon by "two out of three," i.e. it cannot form a regular base pair with the third codon position. In reading the codon CAG the anticodon CUG was approximately eight times more efficient than the anticodon s2UUG. The lysyl-tRNA1Lys (CUU) could not alone sustain any detectable coat protein synthesis in the MS2 system indicating that there was no significant reading of the lysine codon AAA. This conclusion is supported by the outcome of experiments where lysyl-tRNA1Lys (CUU) and lysyl-tRNA2Lys (s2UUU) competed for the codon AAA. The reading efficiency of the anticodon CUU was less than 1% of that of the competing s2UUU which represents the limit of resolution of our experimental system. When the two lysine tRNAs competed for the codon AAG the anticodon CUU was about four times more efficient than s2UUU. These results are discussed in the context of the two out of three hypothesis, which attempts to relate the frequency of such reading to the hydrogen bonding properties of the codon nucleotides.

Bacterial Proteins↗
PubMed1980

Aberrations of the classic codon reading scheme during protein synthesis in vitro.

Using a protein synthesizing in vitro system programmed with MS2-RNA, the ability of alanine tRNAs with the anticodons U*GC (U* represents 5-oxyacetic acid uridine monophosphate) and IGC to read the alanine codons in the coat protein cistron of MS2 has been determined both under conditions of no competition, where the alanyl-tRNA used was the only aminoacylated tRNAAla present in the system, and in experiments where the two alanyl-tRNAs were competing against each other. Under conditions of no competition, each of the anticodons can read all four alanine codons. However, when the anticodons compete for the codon GCC, the anticodon IGC, which can read all three positions of the codon according to the rules of Watson-Crick base pairing, is considerably more efficient than U*GC, which misreads the codon by reading only the first two positions and presumably disregards the third nucleotide of the codon. The outcome of the competition experiments also reveals two apparent violations of the wobble restrictions: the anticodon U*GC reads the codon GUU almost as effectively as does the anticodon IGC, and IGC is almost as effective as U*GC in reading the codon GCG.

Alanine-tRNA Ligase↗
PubMed1979

Relative efficiency of anticodons in reading the valine codons during protein synthesis in vitro.

Using a protein synthesizing in vitro system programmed with MS 2-RNA, the relative efficiency (in the presence of each other) of valine tRNAs with the anticodons U*AC (U* represents 5-oxyacetic acid uridine monophosphate), GAC, and IAC to read the valine codons was investigated. An anticodon which can read all three positions of the codon according to the rules of Watson-Crick base-pairing and the wobble hypothesis is an order of magnitude more efficient than an anticodon which misreads the codon by reading only the first two positions and presumably disregards the third nucleotide of the codon. There are two seeming exceptions to this behavior: the anticodon U*AC reads the codon GUU quite efficiently and IAC is as effective as U*AC in reading the codon GUG. The significance of these exceptions is evaluated with respect to the organization and evolution of the genetic code.

Anticodon↗
PubMed1977

Codon-acticodon recognition in the valine codon family.

An in vitro protein-synthesizing system completely dependent on added valine tRNA (valyl-tRNAval) and programmed with RNA from the phage MS2 has been used to investigate the incorporation into MS2 coat protein of valine from isoaccepting valyl-tRNAsval with the anticodons U AC (U represents 5-oxyacetic acid uridine monophosphate), GAC, and IAC in response to the four valine codons GUU, GUC, GUA, and GUG. By examining the incorporation of valine into NH2-terminal and internal positions of three tryptic peptides from the MS2 coat protein it has been established that these anticodons each recognize all four valine codons. We therefore conclude that under our conditions of in vitro protein synthesis the genetic code, as far as the valine codons are concerned, is operationally a two letter code, i.e. the third codon nucleotide has no absolute discriminating function.

Amino Acid Sequence↗