Phenylalanine Codon Research Articles

Involvement of ribosomal protein L7/L12 in control of translational accuracy.

The effects of two mutations, which map at the rplL locus and both give a changed 50S ribosomal protein L7/L12, were studied. Both mutations are associated with an increased misreading of all three nonsense codons in vivo and ribosomes from the mutants give an increased misreading of the phenylalanine codon UUU by tRNALeu in vitro. The rplL-associated misreading in vitro is not limited to a particular type of mRNA or tRNA. Results from a translational proofreading assay, using mutant ribosomes, suggest that protein L7/L12 is involved in the control of translational accuracy by contributing to the efficiency of a translational proofreading step(s).

Tumor-specific, hypomodified phenylalanyl-tRNA is utilized in translation in preference to the fully modified isoacceptor of normal cells.

Phenylalanine transfer RNA (tRNAPhe) of mammalian tissues contains the hypermodified guanine derivative Y (Wye) adjacent to the 3'-end of the anticodon and two O-methylated bases in the 5' portion of the anticodon loop. These positions are hypomodified in a variety of tumor cells including a mouse neuroblastoma. The normal and tumor-specific Phe-tRNAPhe iso-acceptors were prepared from mouse liver and mouse neuroblastoma cells and compared for their activity in incorporating phenylalanine into each phenylalanine site of rabbit globin in a reticulocyte cell-free protein synthesizing system. The hypomodified Phe-tRNAPhe of neuroblastoma cells is generally preferred to the fully modified tRNAPhe of liver in globin synthesis by about 15%. This preference is the same in the translation of both phenylalanine codons, UUC and UUU, but the ratios of incorporation by the Phe-tRNAPhe species vary from site to site within a 2-fold range. Only 2 of 16 phenylalanine residues are donated preferentially by the fully modified Phe-tRNAPhe. One such residue occurs in beta-42, the second of two tandem phenylalanine residues (both encoded by UUC), while the hypomodified isoacceptor is preferred in translation of the first residue. This result indicates that the translation of tandem residues is particularly affected by the tRNAs available. Since the tumor-specific hypomodified Phe-tRNAPhe is generally utilized preferntially, it appears that the bulky Y base and/or other modifications of normal tRNAPhe may modulate protein synthesis and that tumor cells may achieve a growth advantage if their tRNAPhe is hypomodified.

Comparison of the codon recognition properties and of the utilization of normal and tumor specific Phe-tRNAs in protein synthesis

Phe-tRNA from normal rat liver (designated Phe-tRNA N) and the under-modified, tumor specific, Phe-tRNAs from mouse neuroblastoma (designated Phe-tRNA NB) and rat lymphoma (designated Phe-tRNA RL) recognize the phenylalanine codons, UUU and UUC in a ribosome binding assay, but not other codons that differ from UUU and UUC in a single base at either the 5′ or 3′ position. Phe-tRNA NB was incorporated into protein more extensively than either Phe-tRNA RL or Phe-tRNA N in wheat germ extracts programmed with globin mRNA. The utilization level of each Phe-tRNA was correlated with its rate of deacylation in wheat germ extracts, i.e., Phe-tRNA NB deacylated less rapidly than Phe-tRNA RL or Phe-tRNA N. Phe-tRNA, from which the Y base was chemically excised (designated Phe-tRNA −Y), did not respond to UUU or UUC in the ribosomal binding assay, nor did it transfer its phenylalanine to protein in wheat germ extracts programmed with globin mRNA.

Native and imported transfer RNA in mitochondria.

Tetrahymena mitochondrial and cytoplasmic tRNAs for valine, lysine, arginine and phenylalanine produced identical or different isoacceptor patterns on reversed-phase chromatography. Positive bindings of individual isoacceptors to Escherichia coli ribosomes were demonstrated in response to trinucleoside diphosphates and polynucleotides. It was shown that different isoacceptors exhibited different but degenerate coding recognition patterns. One of two phenylalanyl-tRNA species in mitochondria, however, showed no recognition toward either phenylalanine codon triplets or poly(U). Under suitable annealing conditions, two phenylalanyl-tRNA species could hybridize with mitochondrial DNA. The other mitochondrial tRNAs for valine, lysine and arginine showed no hybridization with mitochondrial DNA but produced significant binding to nuclear DNA-immobilized filters. From these and other coding data presented here, it is postulated that there are two classes of tRNA in Tetrahymena pyriformis mitochondria; the one, “native” tRNA, is mitochondrial DNA-transcript whose coding specificity is unique to mitochondrial tRNA. The other, termed “imported” tRNA, is transcribed from the nuclear DNA and probably transported into mitochondria. This class of tRNA exhibits the same or similar coding specificity as the corresponding cytoplasmic tRNA.

Codon recognition by enzymatically mischarged valine transfer ribonucleic acid.

Direct evidence for the adaptor hypothesis has been obtained by examining the codon recognition of a purified Escherichia coli valine transfer ribonucleic acid which was enzymatically mischarged with phenylalanine labeled with carbn-14 by reaction with purified phenylalanyl-transfer ribonucleic acid synthetase from Neurospora crassa. The mischarged transfer ribonucleic acid recognized the valine codons but failed to recognize the phenylalanine codon when tested in trinucleotide-directed ribosomal binding assay.