Rate-limiting Step In Protein Synthesis Research Articles

Augmentation of Wound Healing with Translation Initiation Factor eIF4E mRNA

Background. Initiation of translation is the rate-limiting step in protein synthesis; eIF4E increases translational efficiency by facilitating ribosome scanning. eIF4E is present in cells in rate-limiting amounts; chronic overexpression of eIF4E causes cell transformation by upregulating growth-related proteins. Biolistic delivery of epidermal growth factor (EGF) increases wound healing; transiently increasing wound eIF4E levels with biolistic mRNA transmission may further augment wound healing without oncogenesis.Patients and methods. Midline fascial wounds were created in rats and biolistically treated with gold particles carrying mRNA encoding for hEGF with or without eIF4E prior to suture closure; control animals received blank bullets. The animals were sacrificed at 7 or 14 days for determination of peak wound bursting strength on a tensiometer. Results are expressed as means ± standard deviation; statistics were via analysis of variance.Results.Conclusions. Simultaneous biolistic delivery of EGF mRNA with eIF4E mRNA significantly increases wound breaking strength compared to that in control animals or treatment with EGF mRNA alone without risk of cellular transformation. Further studies of translational activation to augment wound healing are warranted.

Initiator tRNA and its role in initiation of protein synthesis.

The initiation step of protein synthesis is quite differentfrom the repetitive steps of elongation that follow it. Initiation involves the assembly of the two ribosomal subunits, mRNA, and the initiator tRNA in a reaction requiring the participation of several initiation factors, and isoften the rate-limiting step in protein synthesis (Kozak1983; Gualerzi and Pon 1990; RajBhandary and Chow1995; Hershey and Merrick 2000). The initiator tRNAplays an important role in this process and in the selectionof the appropriate initiation codon. Because of its specialfunction, an initiator tRNA possesses many highly specific properties that are different from those of elongatortRNAs (RajBhandary 1994). We have been interested inidentifying the sequence and/or structural features in theinitiator tRNA important for specifying its distinctiveproperties and in understanding the molecular basis of thespecific interactions of the initiator tRNA with the various proteins, the initiation factors, and the ribosome.Here we report on our work with the eubacterial Escherichia coli initiator tRNA. We describe briefly the systemthat we use for in vitro and in vivo functional analyses ofmutant initiator tRNAs, a summary of the overall results,and some of our conclusions related to participation ofthe tRNA in initiation.

Progressive amplification and overexpression of the eukaryotic initiation factor 4E gene in different zones of head and neck cancers

Purpose: Eukaryotic initiation factor 4E (eIF4E) binds to mRNA as the initial rate-limiting step in protein synthesis. Amplification and overexpression of the eIF4E gene has been associated with malignant transformation. The objectives of this study were to 1) quantify the eIF4E gene in head and neck squamous cell carcinoma (HNSCC) specimens, 2) quantify eIF4E protein elevation and examine its association with eIF4E gene amplification, and 3) determine whether there is progression in eIF4E gene amplification and protein overexpression in the tumor-free resection margin, the transition zone, and the tumor core of HNSCC specimens. Materials and Methods: Eighteen HNSCC specimens were divided into three zones: 1) tumor core; 2) transition zone; and 3) “tumor-free” margin. Competitive polymerase chain reaction was performed to determine eIF4E gene copy number. eIF4E protein expression was quantified using Western blot analysis. Results: All 18 HNSCC specimens tested had significant eIF4E gene amplification (4.3 ± 1.2; P < .05). In contrast, none of the 10 benign specimens from noncancer patients had any eIF4E gene amplification (1.1 ± 0.5). In the 12 HNSCC specimens examined for the three zones, the tumor core and transition zone showed eIF4E gene amplification (5.2 ± 1.1 and 3.5 ± 0.9, respectively) compared with the “tumor-free” margin (2.1 ± 1.1; P < .05). The tumor core and transition zone showed significant eIF4E protein elevation (15.5 ± 9.3, 4.4 ± 4.6, respectively) compared with the “tumor-free” margin (0.9 ± 0.5, P < .05). Conclusions: The eIF4E gene is amplified and overexpressed in HNSCC. Amplification and elevation of eIF4E were highest in the tumor core, intermediate in the transition zone, and lowest in the tumor-free margin. There appears to be progression of eIF4E gene amplification and overexpression from the “tumor-free” margin to the tumor core.

Maintenance of cellular levels of G-proteins: different efficiencies of alpha s and alpha o synthesis in GH3 cells.

G-proteins couple membrane-bound receptors to intracellular effectors. Each cell has a characteristic complement of G-protein alpha, beta and gamma subunits that partly determines the cell's response to external signals. Very little is known about the mechanisms that set and maintain cellular levels of G-proteins or about potential points of regulation. We have assayed the steady-state levels of mRNA and protein for two types of G-protein subunits, alpha s and alpha o, in rat brain, heart and GH3 cells, and found that in all these cases, it takes 9- to 20-fold more mRNA to produce a given amount of alpha s protein than to produce the same amount of alpha o protein. Such a situation could arise from a relatively rapid rate of alpha s protein degradation, requiring rapid protein synthesis to compensate, or from relatively inefficient translation of alpha s mRNA compared with alpha o mRNA. The latter appears to be the case in GH3 cells. These cells contain 94 times more mRNA for alpha s than for alpha o, yet the rate of alpha s protein synthesis is only 9 times greater than alpha o protein synthesis. The degradation rates of the two proteins are similar (13 h for alpha s and 18 h for alpha o). To begin to define the mechanism that accounts for the fact that it takes more mRNA to synthesize a given amount of alpha s than alpha o, we asked whether there is a pool of alpha s mRNA that does not participate in protein synthesis. We found that virtually all alpha s and alpha o mRNA is associated with ribosomes. Therefore, all the mRNA is likely to be capable of directing protein synthesis. Since the rate-limiting step in protein synthesis is usually binding of the ribosome to mRNA at initiation, our results suggest that the relatively slow rate of alpha s protein synthesis is regulated by a mechanism that acts beyond initiation at peptide elongation and/or termination.

Effects of the mechanism of receptor-mediated gene expression on the shape of the dose-response curve.

A mathematical model of receptor-mediated gene expression that includes receptor binding of natural and xenobiotic ligands, protein synthesis and degradation, and metabolism of the xenobiotic ligand was created to identify the determinants of the shape of the dose-response profile. Values of the model's parameters were varied to reflect alternative mechanisms of expression of the protein. These assumptions had dramatic effects on the computed response to a bolus dose of the xenobiotic ligand. If all processes in the model exhibit hyperbolic kinetics, the dose-response curves can appear sigmoidal but actually be linear with a positive slope at low doses. The slope of the curve only approached zero at low dose, indicative of a threshold for response, if binding of the xenobiotic ligand to the receptor exhibited positive cooperativity (ligand binding at one site increases the affinity for ligand at another binding site on the receptor). Positive cooperativity in the rate-limiting step of protein synthesis produced dose-response curves which were "U-shaped" at low doses, also indicative of a threshold. Positive cooperativity in the metabolism of the xenobiotic ligand produced dose-response curves that increased more rapidly than linearly with increasing dose. The model illustrates the fact that response cannot be predicted from qualitative mechanistic arguments alone; any assessment of risk to health from xenobiotic chemicals must be based on a detailed quantitative examination of the kinetic behavior of each chemical species individually.

The role of translocation in ribosomal accuracy. Translocation rates for cognate and noncognate aminoacyl- and peptidyl-tRNAs on Escherichia coli ribosomes.

The ribosomal translocation, as measured in vitro by peptide formation on poly(U)-programmed Escherichia coli ribosomes in the presence of ternary complex, deacylated tRNA or N-acetyl-Phe-tRNA, and elongation factor G, is the rate-limiting step of protein synthesis. Elongation factor G stimulates the spontaneous translocation by a factor of about 500. N-Acetyl-Phe-Phe-tRNA(Phe E. coli) is translocated with a rate constant of 1-2 s-1 at 25 degrees C. Translocation of N-acetyl-Phe-Phe-tRNA(Phe yeast) and N-acetyl-Phe-Leu-tRNA(Leu E. coli) under identical conditions proceeds with a rate by about a factor of 2 and 10, respectively, more slowly. The translocation rate, therefore, is influenced by the nature of the tRNAs in the A-site. We can show, furthermore, that also the tRNA in the P-site, and presumably in the E-site as well, influences the rate of translocation. Reduced rates of translocation of noncognate peptidyl-tRNAs are accompanied by preferential dissociation of these tRNAs at the beginning of the translation of a mRNA.

The effect of N-nitroso carcinogens and triazenes on protein synthesis in cell-free systems

The effect of a series of carcinogenic nitrosamines, alkylnitrosoureas and alkaryltriazenes on enzymatic reactions involved in protein synthesis was studied in cell-free systems from rat liver. The addition of most compounds stimulated the formation of aminoacyl-tRNA complex in test systems from rat liver whereas analogous preparations from Escherichia coli did not show this effect. The polymerization of phenylalanine and the binding of aminoacyl-tRNA to ribosome were only slightly and apparently non-specifically inhibited in the presence of the test compounds. On the other hand, the binding of nRNA to ribosome was markedly stimulated after the addition of most carcinogens tested. It appears that the carcinogens intervene specifically with one of the early steps in peptide initiation. Since the binding of mRNA to ribosome is known to be an important rate-limiting step in protein synthesis, the N-nitroso carcinogens and triazenes may thus control the expression of genetic message at the translation level.

The rate-limiting step of protein synthesis in vivo and in vitro and the distribution of growing peptides between the puromycin-labile and puromycin-non-labile sites on polyribosomes.

1. At 3 min after an intravenous injection of radioactive amino acids into the rat, the bulk of radioactivity associated with liver polyribosomes can be interpreted as growing peptides. 2. In an attempt to identify the rate-limiting step of protein synthesis in vivo and in vitro, use was made of the action of puromycin at 0 degrees C, in releasing growing peptides only from the donor site, to study the distribution of growing peptides between the donor and acceptor sites. 3. Evidence is presented that all growing peptides in a population of liver polyribosomes labelled in vivo are similarly distributed between the donor and acceptor sites, and that the proportion released by puromycin is not an artifact of methodology. 4. The proportion released by puromycin is about 50% for both liver and muscle polyribosomes labelled in vivo, suggesting that neither the availability nor binding of aminoacyl-tRNA nor peptide bond synthesis nor translocation can limit the rate of protein synthesis in vivo. Attempts to alter this by starvation, hypophysectomy, growth hormone, alloxan, insulin and partial hepatectomy were unsuccessful. 5. Growing peptides on liver polyribosomes labelled in a cell-free system in vitro or by incubating hemidiaphragms in vitro were largely in the donor site, suggesting that either the availability or binding of aminoacyl-tRNA, or peptide bond synthesis, must be rate limiting in vitro and that the rate-limiting step differs from that in vivo. 6. Neither in vivo nor in the hemidiaphragm system in vitro was a correlation found between the proportion of growing peptides in the donor site and changes in the rate of incorporation of radioactivity into protein. This could indicate that the intracellular concentration of amino acids or aminoacyl-tRNA limits the rate of protein synthesis and that the increased incorporation results from a rise to a higher but still suboptimum concentration.

Changes in the ribosome distribution during incubation of rabbit reticulocytes in vitro

Intact reticulocytes incubated in vitro synthesize hemoglobin at a linear rate for 1 h at 30°. During the first 30 min about one-third of the 80-S ribosomes are transferred into the polysome fraction, leading to an increase in the concentration and size of polysomes. Protein synthesis is accompanied by polysome breakdown, and by 1 h this is the dominant event. These changes in the ribosome distribution appear to be the result of changes in the initiation process (attachment of ribosomes to polysomes) rather than changes in the rates of peptide bond formation or release of completed polypeptide chains from ribosomes. The rate-limiting step in protein synthesis was located by: (a) determining the synthetic time for the β-chain of hemoglobin within two different time intervals during the incubation, and (b) investigating the kinetics of hemoglobin synthesis under conditions where amino acids were limiting.