Cysteinyl-tRNA Deacylation Can Be Uncoupled from Protein Synthesis

Aminoacyl-tRNA synthetases (ARSs) are critical components of protein translation, providing ribosomes with aminoacyl-tRNAs. In return, ribosomes release uncharged tRNAs as ARS substrates. Here, we show that tRNA deacylation can be uncoupled from protein synthesis in an amino acid specific manner. While tRNAs coupled to radiolabeled Met, Leu Lys, or Ser are stable in cells following translation inhibition with arsenite, radiolabeled Cys is released from tRNA at a high rate. We discuss possible translation independent functions for tRNACys

Synonymous Codon Ordering: A Subtle but Prevalent Strategy of Bacteria to Improve Translational Efficiency

Background: In yeast coding sequences, once a particular codon has been used, subsequent occurrence of the same amino acid tends to use codons sharing the same tRNA. Such a phenomenon of co-tRNA codons pairing bias (CTCPB) is also found in some other eukaryotes but it is not known whether it occurs in prokaryotes. Methodology/Principal Findings: In this study, we focused on a total of 773 bacterial genomes to investigate their synonymous codon pairing preferences. After calculating the actual frequencies of synonymous codon pairs and comparing them with their expected values, we detected an obvious pairing bias towards identical codon pairs. This seems consistent with the previously reported CTCPB phenomenon, since identical codons are certainly read by the same tRNA. However, among co-tRNA but non-identical codon pairs, only 22 were often found overrepresented, suggesting that many co-tRNA codons actually do not preferentially pair together in prokaryotes. Therefore, the previously reported co-tRNA codons pairing rule needs to be more rigorously defined. The affinity differences between a tRNA anticodon and its readable codons should be taken into account. Moreover, both within-gene-shuffling tests and phylogenetic analyses support the idea that translational selection played an important role in shaping the observed synonymous codon pairing pattern in prokaryotes. Conclusions: Overall, a high level of synonymous codon pairing bias was detected in 73 % investigated bacterial species

A channeled tRNA cycle during mammalian protein synthesis.

In earlier studies it was shown that the mammalian translation system is highly organized in vivo and that the intermediates in the process, aminoacyl-tRNAs, are channeled--i.e., they are directly transferred from the aminoacyl-tRNA synthetases to the elongation factor to the ribosomes without dissociating into the cellular fluid. Here, we examine whether spent tRNAs leaving the ribosome enter the fluid phase or are transferred directly to their cognate aminoacyl-tRNA synthetases to complete a channeled tRNA cycle. Using a permeabilized CHO cell system that closely mimics living cells, we find that there is no leakage of endogenous tRNA during many cycles of translation, and protein synthesis remains linear during this period, even though free aminoacyl-tRNA is known to rapidly equilibrate between the inside and outside of these cells. We also find that exogenous tRNA and periodate-oxidized tRNA have no effect on protein synthesis in this system, indicating that they do not enter the translation machinery, despite the fact that exogenous tRNA rapidly distributes throughout the cells. Furthermore, most of the cellular aminoacyl-tRNA synthetases function only with endogenous tRNAs, although a portion can use exogenous tRNA molecules. However, aminoacylation of these exogenous tRNAs is strongly inhibited by oxidized tRNA; this inhibitor has no effect on endogenous aminoacylation. On the basis of these and the earlier observations, we conclude that endogenous tRNA is never free of the protein synthetic machinery at any stage of the translation process and, consequently, that there is a channeled tRNA cycle during protein synthesis in mammalian cells

Supramolecular organization of the mammalian translation system.

Although evidence suggests that the protein synthetic machinery is organized within cells, this point has been difficult to prove because any organization that might exist is lost upon preparation of the cell-free systems usually used to study translation in vitro. To examine this process under conditions more representative of the intact cell, we have developed an active protein-synthesizing system using Chinese hamster ovary (CHO) cells permeabilized with the plant glycoside saponin. This procedure renders cells permeable to trypan blue and exogenous tRNA, but there is little release of endogenous macromolecules. Protein synthesis in this system proceeds at the same rate as that in intact cells and is about 40-fold faster than that in a cell-free system prepared from the same cells. Active protein synthesis in this system requires the addition of only Mg2+, K+, and creatine phosphate, with a small further stimulation by ATP and an amino acid mixture; no exogenous macromolecules are necessary. The proteins synthesized in this system are indistinguishable from those made by the intact cell, and the channeling of aminoacyl-tRNA observed in vivo is maintained. Our data suggest that the permeabilized cell system retains the protein-synthesizing capabilities of the intact cell and presumably its internal structure as well. Studies with this system demonstrate that the protein-synthesizing apparatus is highly organized and that its macromolecular components are not freely diffusible in mammalian cells

Reduced attachment of blood mononuclear and alphaM-beta2-expressing cells to surfaces coated with fish proteins

Abstract not available

Fish protein coatings that reduce bacteria and macrophage attachment to surfaces

Abstract not available

Incorporation of Lysyl-tRNA Synthetase into Human Immunodeficiency Virus Type 1

During human immunodeficiency virus type 1 (HIV-1) assembly, tRNA(Lys) isoacceptors are selectively incorporated into virions and tRNA [Formula: see text] is used as the primer for reverse transcription. We show herein that the tRNA(Lys)-binding protein, lysyl-tRNA synthetase (LysRS), is also selectively packaged into HIV-1. The viral precursor protein Pr55(gag) alone will package LysRS into Pr55(gag) particles, independently of tRNA(Lys). With the additional presence of the viral precursor protein Pr160(gag-pol), tRNA(Lys) and LysRS are both packaged into the particle. While the predominant cytoplasmic LysRS has an apparent M(r) of 70,000, viral LysRS associated with tRNA(Lys) packaging is shorter, with an apparent M(r) of 63,000. The truncation occurs independently of viral protease and might be required to facilitate interactions involved in the selective packaging and genomic placement of primer tRNA [Formula: see text]