Showing papers by "Murray P. Deutscher published in 1992"
TL;DR: Site-directed mutagenesis of Escherichia coli gene encoding RNase D has shown that a U8 sequence upstream of the Shine-Dalgarno region is also essential for expression of this mRNA, demonstrating that sequences other than those previously recognized can be essential for translation initiation.
Abstract: Binding of 30S ribosomal subunits to mRNA during the initiation of prokaryotic translation is known to be influenced by the initiation codon and the Shine-Dalgarno sequence Site-directed mutagenesis of rnd, the Escherichia coli gene encoding RNase D, has now shown that a U8 sequence upstream of the Shine-Dalgarno region is also essential for expression of this mRNA Alteration of two to five uridine residues within this sequence has no effect on mRNA levels but decreases RNase D protein and activity by as much as 95%, indicating that the U-rich sequence acts as an enhancer of translation Moreover, mutant transcripts bind to 30S ribosomes in vitro with lower affinity than their wild-type counterparts, suggesting that the role of the U8 sequence is in the initial binding of ribosomes to the translation initiation region of the message These data demonstrate that sequences other than those previously recognized can be essential for translation initiation
76 citations
TL;DR: The data indicate that these five exoribonucleases functionally overlap in vivo and that any one of them can take over the functions of all the others, although with various efficiencies.
Abstract: Escherichia coli contains multiple exoribonucleases. Strains lacking the exoribonucleases RNase II, D, BN, T, and PH are inviable. The introduction of a chromosomal, wild-type copy of the gene for any one of these enzymes is sufficient to allow cell growth, with the enzymes being in the following order of effectiveness: RNase T > RNase PH > RNase D > RNase II > RNase BN. The data indicate that these five exoribonucleases functionally overlap in vivo and that any one of them can take over the functions of all the others, although with various efficiencies.
70 citations
TL;DR: It is concluded that aminoacyl-tRNAs synthesized in vivo are continually associated with components of the protein synthesis machinery and are thereby retained within the permeabilized cell and are also protected from RNase action.
Abstract: We have recently proposed that aminoacyl-tRNA is channeled during protein synthesis in vivo--i.e., it is directly transferred among the components of the protein-synthesizing machinery and does not mix with aminoacyl-tRNA molecules introduced from outside the cell. To understand the structural basis for these functional properties, we have examined the disposition of aminoacyl-tRNA within the cell. To do this we have developed a Chinese hamster ovary (CHO) permeabilized-cell system using the plant glycoside saponin. We show, using a mixture of free 14C-labeled amino acids and 3H-labeled aminoacyl-tRNAs, that 14C-labeled aminoacyl-tRNAs synthesized endogenously from the free amino acids are preferentially sequestered within the cell, whereas their exogenous 3H counterparts distribute between the inside and outside of the cell based solely on the relative volumes of the two compartments. Furthermore, the endogenous 14C-labeled aminoacyl-tRNA population is resistant to pancreatic ribonuclease action, whereas the 3H molecules are rapidly degraded. We conclude, based on these observations, that aminoacyl-tRNAs synthesized in vivo are continually associated with components of the protein synthesis machinery and are thereby retained within the permeabilized cell and are also protected from RNase action. These data provide independent evidence for the channeling model of protein biosynthesis.
64 citations
TL;DR: The OrfE protein has both the degradative and synthetic activities of RNase PH, and the equilibrium constant for RNasePH is near unity, suggesting that at the phosphate concentration present in vivo, the enzyme would participate in RNA degradation.
57 citations
TL;DR: In an in vitro tRNA processing system the defect caused by elimination of RNase PH was shown to be the accumulation of a precursor that contained 4-6 additional 3' nucleotides following the -CCA sequence, indicating that RNasePH can be an essential enzyme for the processing of tRNA precursors.
51 citations
TL;DR: The Escherichia coli rnt gene encoding the enzyme RNase T, which is responsible for the end-turnover of tRNA, was cloned on a 1.5-kilobase DNA fragment and primer extension analysis revealed that the rnt message also included the second open reading frame, suggesting that rnt is part of an operon.
27 citations
TL;DR: A clone containing the Escherichia coli rna gene encoding the nonspecific endoribonuclease, RNase I, was isolated and sequenced, and results indicate that the rna promoter is highly unusual.
11 citations