The Effect of Salt Concentration on the Inhibition of Protein Synthesis by Double-Stranded RNA (original) (raw)
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THE NUMBER OF SOLUBLE RNA MOLECULES ON RETICULOCYTE POLYRIBOSOMES
Proceedings of the National Academy of Sciences, 1964
During the past few years, wie have come to know the general outline of the mechanism of protein synthesis. On the ribosomes, the polypeptide chain grows from one end' by the stepwise addition of amino acids carried by soluble RNA (sRNA).A It is believed that the sequence of amino acids is determined by the sequence of nucleotides in the associated messenger RNA.3' Several workers have studied the in vitro binding of sRNA to ribosomes.5'-0 Takanami,7 as well as Cannon et al.,9 has found that one sRNA molecule binds to purified ribosomes. On the basis of this analysis, Gilbert11 has suggested a mode of action of sRNA on the ribosome during protein synthesis. However, it would be desirable to have a direct analysis of the number of sRNA molecules associated with each ribosome in the intact cell. In the work reported here, we have used the specialized properties of the reticulocyte to make such an analysis of ribosomes which are active in protein synthesis.
European Journal of Biochemistry, 1978
Rabbit reticulocyte lysates preincubated at 42 -45 "C and subsequently assayed for protein synthesis at 37 "C show (a) a decrease in their rate of polypeptide chain initiation and (b) decreased Met-tRNAf binding to native 40-S subunits and 80-S ribosomes. Similarly, the amount of [3sS]Met-tRNAf . 40-S-subunit initiation complexes formed in vitro with native 40-S subunits isolated from supraoptimally heated lysates is only 10 -30 of that formed with native 40-S subunits prepared from control lysates preincubatedat 0 "C or 37 "C. This decrease is not due to reduced formation of Met-tRNAr ternary complex or to increased destruction of this complex, since we find that 0.5 M KC1 ribosomal washes extracted from lysates preincubated at 0 "C, 37 "C, or 43-45 C have almost identical activities in Met-tRNAf . GTP . eIF-2 ternary complex formation and Met-tRNAf hydrolysis, whether assayed at 37 "C or 45 "C. Similarly, the results of binding assays of [35S]Met-tRNAf to 40-S subunits performed at both temperatures show that the control and heated washes, added at either limiting or optimal amounts, stimulate almost equally the binding of Met-tRNAf to (a) salt-washed derived 40-S subunits and (b) native 40-S subunits, prepared from control or supraoptimally heated lysates. Moreover the factor-directed binding of Met-tRNAf by the derived 40-S subunits is not affected by the presence of either the control or heated postribosomal supernatant. We conclude that at elevated temperatures there is no irreversible inactivation of the Met-tRNAf binding factor for ternary complex formation and Met-tRNAf binding to 40-S subunits in vitro and discuss some possible explanations for the lowered Met-tRNAf binding observed in vivo.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1986
Inhibition of polypeptide chain initiation in rabbit reticulocyte lysate by phosphorylation of eukaryotic initiation factor-2(a) results, secondarily, in the enzymatic deacylation of Met-tRNAf on the 48 S initiation complexes that accumulate. We have prepared an antibody to a highly purified preparation of the major Met-tRNAf deacylase activity on rabbit reticulocyte ribosomes, termed deacylase II. Antibody, but not similarly purified normal IgG, completely neutralizes the activity of Met-tRNAf deacylase II and has no effect on Met-tRNAf deacylase I, a separate, minor, reticulocyte activity with the same substrate specificity but very different physical and enzymatic properties, strongly suggesting that deacylase I and II are distinct proteins. We partially purified Met-tRNAf deacylase activities from rabbit liver, myocardium and bone marrow ribosomes and found them to be similar to each other and to reticulocyte deacylase I in their enzymatic properties and insensitivity to anti-deacylase II, suggesting that deacylase I may be a general form of this enzyme, present in many cells, while deacylase II may be induced specifically during erythroid differentiation. Addition of the antibody to reticulocyte iysate incubated in the absence of hemin or presence of hemin plus 0.1 ttg/ml poly(l-C) did not reverse the inhibition of protein synthesis but did reduce the rate of turnover/utilization of Met-tRNA r and increase the level of Met-tRNAf bound to 48 S initiation complexes, demonstrating that the deacylase does not directly inhibit protein synthesis under these conditions but does mediate the deacylation, loss, and thus greater than expected turnover of Met-tRNA f in the 48 S complexes that accumulate.
Protein synthesis in rabbit reticulocytes: Characteristics of a Met-tRNAfMet binding factor
Biochemical and Biophysical Research Communications, 1972
Three factors (IFl, IF*, IFS) resolved by DEAE-cellulose column chromatography of the crude reticulocyte ribosomal salt wash (I fraction), Met stimulated poly r(U-G) directed methionine transfer from Met-tRNAf into the terminal positions of the synthesized polypeptides. All three factors were necessary for maximum methionine transfer. A protein factor that is also present in the I fraction and elutes Met similarly to IF1 on DEAE-cellulose chromatography, binds Met-tRNAf in the presence of GTP. The complex formed is quantitatively retained on Millipore filter. Met This complex formation is specific for Met-tRNAf. Other amino acyl tRNAs tested such as Met-'RNA, Phe Met, Phe-tRNA and Val-tRNAVal do not Met form a similar complex. With Met-tRNAf , complex formation does not require Met tion that binds specifically Met-tRNAf in the presence of GTP. The complex Copyright 0 1972 by Academic Press. Inc. All rights of reproduction i n my form reserved. formation is assayed by its retention on Millipore filter. This complex formation does not require Mg +t ion, and is inhibited by the addition of Public Health Service Career Development Award (l-K4-GM-46, 240-01) to N.K.G.
Met-tRNA hydrolase from reticulocytes specific for on 40S ribosomal subunits
Archives of Biochemistry and Biophysics, 1972
A protein fraction removed from rabbit reticulocyte ribosomes by 0.5 M KC1 releases methionine from methionyl-tRNAr Met bound to the smaller ribosomal subunit. The active component appears to be a Met-tRNA hydrolase. In the presence of the smaller subunit, the hydrolysis reaction is dependent upon codon-directed binding. Release of methionine from methionyl-tRNAtMet is inhibited by addition of larger ribosomal subunits to the reaction mixture. Both Met-tRNA#et and Met-tRNAfMet are hydrolyzed at low Mg* concentration in the absence of 405 subunits, however, the relative hydrolysis of Met-tRNAnMet is reduced in the presence of subunits. The Met-tRNAfMet binding requirement for hydrolysis in the presence of the subunits appears to reflect binding of the enzyme to the smaller subunit. Little or no hydrolysis is observed with N-acetylmethionyl-tRNAfn"t,
European Journal of Biochemistry, 1978
A factor isolated from rabbit reticulocyte white ghosts by Triton X-100 treatment blocks protein synthesis at the elongation-termination stage. Factor-treated ribosomes were found to have an identical buoyant density to that of control ribosomes. When incubated with either reticulocyte ribosomes or ribosomal RNA, the factor products specific cuts in the 28-S ribosomal RNA component without damaging the 18-S RNA. Incubations of pancreatic or TI RNase, with ribosomal RNA, at similar protein-synthesis inhibitory concentrations effected a complete breakdown to oligo and mononucleotides.
Binding of aminoacyl transfer ribonucleic acid synthetases to ribosomes from rabbit reticulocytes
Biochemistry, 1972
Some, but not all, of the aminoacyl-tRNA synthetases present in lysates of rabbit reticulocytes are recovered in high proportion with ribosomes collected by high-speed centrifugation. Up to 90% of the phenylalanyl-tRNA synthetase activity in the lysate is recovered in the ribosomal fraction and appears to have been physically bound to the ribosomes in intact cells. Synthetase activity for lysine and arginine is bound to the ribosome but also appears as a high W e have reported previously the existence of a factor in the salt-wash fraction from reticulocyte ribosomes which promotes binding of N-AcPhe-tRNA or Phe-tRNA to nitrocellulose filters or to ribosomes (McKeehan et al., 1970). This factor was identified as Phe-tRNA synthetase on the basis of cochromatography of the activity for binding and the synthesis of Phe-tRNA plus the specific inhibitory effect of phenylalanine and ATP on N-AcPhe-tRNA binding. It was concluded that a phenylalanyl adenylate complex was formed on Phe-tRNA synthetase bound to ribosomes. Here, we extend these studies and demonstrate an interaction of other synthetases with ribosomes.