Initiation Of Polypeptide Synthesis Research Articles

A rapid purification protocol for the mitogen-activated p70 S6 kinase.

Reentry into the cell cycle from quiescence is stimulated by mitogens and supported by increased levels of protein synthesis. Initiation of polypeptide synthesis is the step most often subject to regulation. This is controlled by phosphorylation of a number of initiation factors and the single 40S ribosomal protein S6. The kinase responsible for S6 phosphorylation is p70(S6k). In the past, the apparent low cellular abundance of p70(S6k) resulted in the development of laborious large-scale purification procedures. Here a rapid and low-cost protocol which yields p70(S6k) purified to near homogeneity and with high specific activity is described. With respect to previous strategies, this novel scheme takes advantage of recently improved ion-exchange media in the first and second purification steps, thus allowing the effective removal of bulk proteins and the major Ser/Thr phosphatases. The third step of purification consists of a single affinity chromatography column in which the ligand is a 20-residue peptide containing the structural motif required for recognition and binding by p70(S6k). This novel protocol allows the rapid purification of large amounts of p70(S6k) and will facilitate the screening of libraries of natural or synthetic compounds aimed at the identification of p70(S6k) inhibitory molecules.

The Phosphodiesterase Inhibitor SQ 20006 Selectively Blocks Mitogen Activation of p70S6k and Transition to S Phase of the Cell Division Cycle without Affecting the Steady State Phosphorylation of eIF-4E

In quiescent cells high levels of protein synthesis are required in order to re-enter the cell cycle upon stimulation. Initiation of polypeptide synthesis is the step most often subject to regulation, controlled in part by phosphorylation of 40 S ribosomal protein S6 and a number of initiation factors. The kinase responsible for S6 phosphorylation is p70S6k. We now show that the p70S6k pathway can be selectively blocked by the aminopurine analogue, SQ 20006. This agent is known to raise cAMP levels, resulting in activation of protein kinase A. We present evidence that the increase in cAMP is not responsible for the inhibitory effect observed. We also show that SQ 20006 can prevent the activation of p70S6k in a rapid and reversible manner. The compound does not exert its inhibitory activity on p70S6k but can inhibit in vitro two protein kinase C isozymes (alpha and gamma). In a B lymphoblastoid cell line, treatment with SQ 20006 results in inhibition of protein synthesis at the initiation stage. In contrast, when tested directly upon the translational machinery in the reticulocyte lysate, inhibition is manifest at both the level of initiation and elongation. The role of protein kinase A in the modulation of p70S6k and the rate of translation is discussed.

ATPase activities of wheat germ initiation factors 4A, 4B, and 4F.

Eukaryotic initiation factors (eIF)-4A, -4B, and -4F isolated from wheat germ were tested for their ability to catalyze ATP hydrolysis in the absence and presence of RNA. We find that eIF-4B or eIF-4F alone exhibit ATPase activity in the presence of poly(U), satellite tobacco necrosis virus (STNV) RNA, or globin mRNA but not in the absence of RNA. eIF-4A alone exhibits ATPase activity in the absence of RNA, but this activity is not stimulated by the addition of RNA. eIF-4A does, however, enhance RNA-dependent ATP hydrolysis in the presence of either eIF-4B or eIF-4F. The RNA-dependent ATPase activities of eIF-4B and eIF-4F are additive, not synergistic. At saturating concentrations of eIF-4F, little or no stimulation of ATP hydrolysis is obtained upon the addition of eIF-4B and at saturating concentrations of eIF-4B little or no stimulation is obtained upon the addition of eIF-4F. This observation is in agreement with our previous finding (Lax, S., Fritz, W., Browning, K., and Ravel, J. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 330-333) that initiation of polypeptide synthesis is obtained in vitro with either eIF-4F or eIF-4B.

Identification of herpesvirus sylvilagus-induced polypeptides in productively infected cells.

Polypeptides synthesized in cell cultures infected with high multiplicities of herpesvirus sylvilagus were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled cell extracts. Initiation of polypeptide synthesis was detected by 6 h after infection. The maximum intensity of many [35S]methionine-labeled viral bands was observed at 45 h after infection. Production of detectable infectious virus began between 18 and 24 h and reached a plateau at 48 h after infection. Immunoprecipitation of cell extracts identified a minimum of 45 virus-induced polypeptides ranging in molecular weight from 230,000 to 27,000. The major polypeptide appeared to have a molecular weight of 150,000. The pattern of these extracts suggested that the synthesis of host polypeptides is stimulated during the first 12 h and thereafter reduced, but not completely inhibited, during the remaining course of infection.

Hormone-mediated translational control of protein synthesis in cultured cells of Glycine max

Proliferative growth in cultured soybean cells ( Glycine max cv. Sodifuri, of cotyledenary origin) is regulated to a substantial degree by the plant hormone, cytokinin. Previously we reported that stationary phase soybean cells exhibit a rapid increase in polyribosome formation upon transfer to fresh cytokinin-containing medium. Cytokinin plays a significant role in this expansion of the cell's protein synthetic capacity through a heretofore unidentified effect on protein synthesis at the translational level. The present study was undertaken to identify the deficiency in the translational process of stationary phase cells which is overcome by cytokinin treatment. We have demonstrated that cytokinin-induced polyribosome formation was brought about by the recruitment of existing monoribosomes into polyribosomal aggregates. Three hours of cytokinin stimulation nearly doubled the total amount of [ 3H]leucine incorporated into nascent polypeptides. Average polyribosome size was nearly identical in stationary phase cells and in cells which were induced to grow by cytokinin. Cytokinin-enhanced protein synthetic capacity was not accompanied by changes in polypeptide chain elongation or termination rates. Partial cycloheximide inhibition of elongation was used to demonstrate that initiation of polypeptide synthesis was limiting to a slight degree both before and after cytokinin stimulation. These results suggest that cytokinin stimulates protein synthesis by making more message available for translation. We obtained direct evidence to support this hypothesis by showing that labeled poly(A)-containing, nonpolyribosomal RNA, which was synthesized during stationary phase but not translated, moves into polyribosomes upon cytokinin treatment.

Initiation of polypeptide synthesis with various NH2-blocked aminoacyl-tRNAs under the direction of alfalfa mosaic virus RNA 4.

Initiation of polypeptide synthesis in a cell-free system of Escherichia coli directed by alfalfa mosaic virus RNA 4 was studied by using either fMet-tRNA or Ac-Phe-tRNA as initiator tRNA. Initiation with fMet-tRNA yielded a product that was identical to the authentic viral coat protein except that the NH2-terminal serine was preceded by fMet instead of being acetylated. When Ac-Phe-tRNA was used as initiator, the biosynthetic product was 10-12 amino acid residues longer, the extra amino acids being located at the NH2-terminus. fMet-tRNA and Ac-Phe-tRNA did not compete for ribosomes during initiation of protein synthesis, as became evident from incorporation studies using both initiator tRNAs simultaneously. It is concluded that E. coli ribosomes recognize two sites on the 5' end of alfalfa mosaic virus RNA 4 that are separated by a region of about 30 nucleotides. The results are in complete agreement with the 5'-terminal nucleotide sequence of this RNA [Koper-Zwarthoff, E. C., Lockhard, R. E., RajBhandary, U. L., Alzner-deWeerd, B. & Bol, J. F. (1977) Proc. Natl. Acad. Sci. USA 74, 5504-5508].

On the identification of the two E. coli supernatant proteins which stimulate the initiation of polypeptide synthesis

The two E. coli supernatant proteins which were previously designated as factors A and B, and which were shown to activate ribosomes for initiation, are very probably factors EF-Ts and EF-G, respectively. An EF-Ts preparation, homogeneous when analyzed by polyacrylamide gel electrophoresis, contained considerable A activity. The EF-Ts and A activities differed, however, in their heat sensitivity and in the amount of the preparation required. An electrophoretically-homogeneous preparation of EF-G retained strong B activity. The amount of the preparation required for EF-G and B activity and the heat sensitivity of the two activities were similar. GTP enhanced the activation of the ribosomes by factors A and B.

Initiation of synthesis of N-terminal acetylated histones with methionine.

The studies presented were undertaken to clarify the unsettled question whether the synthesis of histones with a N-acetylserine residue at the amino terminal end is initiated with methionine. Histones were synthesized in vitro in a rabbit reticulocyte lysate, primed with a mRNA preparation from ascites cells. Initiation of polypeptide synthesis was investigated by using N-formyl[35S]met-tRNAfMet from yeast to label the N-termini. N-Formylmethionine was incorporated into histones H1 and H4 whose N-terminal amino acid is alpha-N-acetylserine. By comparison of tryptic peptides derived from these two histones labeled either with methionine or formylmethionine and from Edman degradation it is shown that N-terminal acetylated histones are initiated with methionine, as is the case for other eukaryotic and bacterial proteins.

Requirement of initiation factor 3 in the initiation of polypeptide synthesis with N-acetylphenylalanyl-tRNA.

Initiation factor 3 is required, along with initiation factors 1 and 2, for the incorporation of N-acetylphenylalanine into polypeptides and the formation of N-acetylphenylalanylpuromycin. Initiation factor 3 also strongly stimulates the binding of N-acetylphenylalanyl transfer RNA to isolated 30S ribosomal subunits. Phosphocellulose fractions of initiation factor 3 were found to catalyze N-acetylphenylalanine incorporation differentially with different synthetic messenger RNAs not containing any codons for N-formylmethionine. The results suggest that ribosomes recognize the initiator codon only through the initiator transfer RNA.

Osmoregulation of ribosomal function in mouse liver

It has been demonstrated earlier that the administration orally or intraperitoneally of hypertonic solutions causes disaggregation of polyribosomes and inhibition of protein synthesis in the livers of mice. In this study, purified hepatic ribosomes were tested in vitro for their activity in initiation of protein synthesis. Ribosomes which were obtained from mice that received intraperitoneally a 4 % NaCl (hypertonic) solution were less active in initiation of polypeptide synthesis than those obtained from control mice that received a 0.7 % NaCl (isotonic) solution. Initiation factors prepared from livers of both groups were equally active. These results offer some insight into the mechanism by which hyperosmolarity of portal blood induces disaggregation of hepatic polyribosomes.

Chain initiation during polypeptide synthesis in cell-free bacterial systems programmed with a plant viral messenger: Initiation with N-acetylated aminoacyl-tRNAs on adjacent codons

As a part of a more extensive program dealing with the initiation of polypeptide synthesis in cell-free systems of Escherichia coli directed by plant viral RNAs and bacteriophage RNA, the role of N-acetylated aminoacyl-tRNAs in this process has been studied. The viral messenger was RNA derived from the top component a of alfalfa mosaic virus (AMV-RNA). The primary interaction between AMV-RNA and E. coli ribosomes is very selective. Only three species of aminoacyl-tRNA: phenylalanyl-tRNA, isoleucyl-tRNA, and valyl-tRNA can bind to the ribosome-AMV-RNA complex. Two of these species, when N-acetylated, can function as polypeptide chain initiator: N-acetylphenylalanyl-tRNA and N-acetylisoleucyl-tRNA. By contrast N-acetylvalyl-tRNA does not bind to the ribosome-mRNA complex and cannot act as chain initiator either. Analysis of the biosynthetic product formed with N-acetyl- 14C-phenylalanyl-tRNA revealed that N-acetylphenylalanine is incorporated into the N-terminal structure of the product. Deacetylation appeared to be very limited under the conditions of incorporation. Aminoacyl-tRNA species, which might respond to codons overlapping the two adjacent ones in the hexanucleotide coding for phenylalanine and isoleucine, do not bind to the mRNA-ribosome complex and presumably cannot function as chain initiator when N-acetylated. This has been confirmed experimentally for N-acetylleucyl-tRNA. Ribosomal initiation factors and GTP, which at low Mg 2+ concentration are essential for binding of N-acetylphenylalanyl-tRNA to the ribosomes programmed with poly U, do not promote the binding of the tRNA or N-acetylisoleucyl-tRNA to the AMV-RNA-ribosome complex. It is concluded that chain initiation in the presence of AMV-RNA can occur by means of two different mechanisms: one requires low Mg 2+ concentrations, initiation factors, GTP and N-formylmethionyl-tRNA (F-met-tRNA); the other can make use of N-acetylaminoacyl-tRNAs at relatively high Mg 2+ concentrations in the absence of initiation factors and GTP. The implications in connection with correct translation are discussed.

A possible role of the AUG codon in the initiation of polypeptide synthesis in a eukaryotic organism

A possible role of the AUG codon in the initiation of polypeptide synthesis in a eukaryotic organism

Regulation of protein synthesis in mammalian cells: II. Inhibition of protein synthesis at the level of initiation during mitosis

The decline in the rate of protein synthesis during mitosis in eukaryotic cells was studied using Chinese hamster ovary cells arrested in metaphase with Colcemid. The rate of protein synthesis in mitotic cells is approximately 30% of the interphase rate. Ribosomes become attached to messenger RNA and initiate polypeptide synthesis at a reduced rate. After initiation of polypeptide synthesis, ribosomes are translated at a normal rate. Reducing the rate of translation with low levels of cycloheximide mobilizes a large proportion of monomeric ribosomes into active polyribosomes. The amount of messenger RNA associated with polyribosomes is also increased in mitotic cells treated with cycloheximide. It appears that the reduced protein synthesis during mitosis results from a lowered rate of attachment of ribosomes to messenger RNA and initiation of polypeptide synthesis.

In vitro protein synthesis directed by R17 viral ribonucleic acid. I. Initiation of polypeptide synthesis in a purified cell-free system.

In vitro protein synthesis directed by R17 viral ribonucleic acid. I. Initiation of polypeptide synthesis in a purified cell-free system.