Rate Of Polypeptide Chain Elongation Research Articles

Formyl-methionine as a degradation signal at the N-termini of bacterial proteins.

In bacteria, all nascent proteins bear the pretranslationally formed N-terminal formyl-methionine (fMet) residue. The fMet residue is cotranslationally deformylated by a ribosome-associated deformylase. The formylation of N-terminal Met in bacterial proteins is not strictly essential for either translation or cell viability. Moreover, protein synthesis by the cytosolic ribosomes of eukaryotes does not involve the formylation of N-terminal Met. What, then, is the main biological function of this metabolically costly, transient, and not strictly essential modification of N terminal Met, and why has Met formylation not been eliminated during bacterial evolution? One possibility is that the similarity of the formyl and acetyl groups, their identical locations in N terminally formylated (Nt formylated) and Nt-acetylated proteins, and the recently discovered proteolytic function of Nt-acetylation in eukaryotes might also signify a proteolytic role of Nt formylation in bacteria. We addressed this hypothesis about fMet based degradation signals, termed fMet/N-degrons, using specific E. coli mutants, pulse-chase degradation assays, and protein reporters whose deformylation was altered, through site-directed mutagenesis, to be either rapid or relatively slow. Our findings strongly suggest that the formylated N-terminal fMet can act as a degradation signal, largely a cotranslational one. One likely function of fMet/N-degrons is the control of protein quality. In bacteria, the rate of polypeptide chain elongation is nearly an order of magnitude higher than in eukaryotes. We suggest that the faster emergence of nascent proteins from bacterial ribosomes is one mechanistic and evolutionary reason for the pretranslational design of bacterial fMet/N degrons, in contrast to the cotranslational design of analogous Ac/N degrons in eukaryotes.

Nanosecond Dynamics of Calmodulin and Ribosome‐Bound Nascent Chains Studied by Time‐Resolved Fluorescence Anisotropy

We report a time-resolved fluorescence anisotropy study of ribosome-bound nascent chains (RNCs) of calmodulin (CaM), a prototypical member of the EF-hand family of calcium-sensing proteins. As shown in numerous studies, in vitro protein refolding can differ substantially from biosynthetic protein folding, which takes place cotranslationally and depends on the rate of polypeptide chain elongation. A challenge in this respect is to characterize the adopted conformations of nascent chains before their release from the ribosome. CaM RNCs (full-length, half-length, and first EF-hand only) were synthesized in vitro. All constructs contained a tetracysteine motif site-specifically incorporated in the first N-terminal helix; this motif is known to react with FlAsH, a biarsenic fluorescein derivative. As the dye is rotationally locked to this helix, we characterized the structural properties and folding states of polypeptide chains tethered to ribosomes and compared these with released chains. Importantly, we observed decelerated tumbling motions of ribosome-tethered and partially folded nascent chains, compared to released chains. This indicates a pronounced interaction between nascent chains and the ribosome surface, and might reflect chaperone activity of the ribosome.

Chronic improvement of amino acid nutrition stimulates initiation of global messenger ribonucleic acid translation in tissues of sheep without affecting protein elongation1

Initiation of mRNA translation and elongation of the polypeptide chain are 2 regulated processes responsible for the short-term postprandial acceleration of protein synthesis in animal tissues. It is known that a chronic increase in the absorptive supply of AA stimulates protein synthesis in ruminant animals, but effects on translation initiation and elongation are unknown. To determine whether initiation or elongation phases of global mRNA translation are affected by chronic elevation of AA supply, 24 ewe lambs of 25.9 +/- 2.5 kg of BW were randomly allocated to 4 treatment groups of 6 lambs each. All lambs received a basal diet of barley and hay at 1.2 times maintenance ME intake. Treatments were an intravenous (i.v.) saline infusion as a control, i.v. infusion of 6 essential AA (EAA; Arg, Lys, His, Thr, Met, Cys) for 10 d, i.v. infusion of the same EAA excluding Met and Cys (EAA-SAA) for 10 d, and an oral drench of fishmeal twice daily for 17 d. Fishmeal supplementation supplied an extra 719 mg of N x kg(-0.75) x d(-1) and N retention was increased 519 mg x kg(-0.75) x d(-1) over the control. The EAA treatment supplied an extra 343 mg of N x kg(-0.75) x d(-1) directly into the blood, and N balance was increased by 268 mg x kg(-0.75) x d(-1). Deletion of Met plus Cys from EAA had no effect on N balance. The results indicate that Met plus Cys did not limit body protein gain on the basal diet alone or the basal diet plus 6 AA. Protein fractional synthesis rates in liver, duodenum, skin, rumen, semimembranosus, and LM were measured by a flooding dose procedure using L-[ring-2,6-(3)H]-Phe. Ribosome transit times were estimated from the ratio of nascent to total protein-bound radioactivities. Fishmeal and EAA treatments had no effect on RNA, DNA, or protein contents of tissues, but fractional synthesis rate, translational efficiency, and concentrations of active ribosomes were consistently elevated. Ribosome transit time was not affected by long-term AA supply. We conclude that the chronic stimulation of protein synthesis by long-term i.v. infusion of EAA or supplementation with an undegradable protein source is brought about by an improvement in the rate of initiation of mRNA translation with no change in the rate of polypeptide chain elongation.

Mapping the functional domains of elongation factor-2 kinase.

A new class of eukaryotic protein kinases that are not homologous to members of the serine/threonine/tyrosine protein kinase superfamily was recently identified [Futey, L. M., et al. (1995) J. Biol. Chem. 270, 523-529; Ryazanov, A. G., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 4884-4889]. This class includes eukaryotic elongation factor-2 kinase, Dictyostelium myosin heavy chain kinases A, B, and C, and several mammalian putative protein kinases that are not yet fully characterized [Ryazanov, A. G., et al. (1999) Curr. Biol. 9, R43-R45]. eEF-2 kinase is a ubiquitous protein kinase that phosphorylates and inactivates eukaryotic translational elongation factor-2, and thus can modulate the rate of polypeptide chain elongation during translation. eEF-2 was the only known substrate for eEF-2 kinase. We demonstrate here that eEF-2 kinase can efficiently phosphorylate a 16-amino acid peptide, MH-1, corresponding to the myosin heavy chain kinase A phosphorylation site in Dictyostelium myosin heavy chains. This enabled us to develop a rapid assay for eEF-2 kinase activity. To localize the functional domains of eEF-2 kinase, we expressed human eEF-2 kinase in Escherichia coli as a GST-tagged fusion protein, and then performed systematic in vitro deletion mutagenesis. We analyzed eEF-2 kinase deletion mutants for the ability to autophosphorylate, and to phosphorylate eEF-2 as well as a peptide substrate, MH-1. Mutants with deletions between amino acids 51 and 335 were unable to autophosphorylate, and were also unable to phosphorylate eEF-2 and MH-1. Mutants with deletions between amino acids 521 and 725 were unable to phosphorylate eEF-2, but were still able to autophosphorylate and to phosphorylate MH-1. The kinases with deletions between amino acids 2 and 50 and 336 and 520 were able to catalyze all three reactions. In addition, the C-terminal domain expressed alone (amino acids 336-725) binds eEF-2 in a coprecipitation assay. These results suggest that eEF-2 kinase consists of two domains connected by a linker region. The amino-terminal domain contains the catalytic domain, while the carboxyl-terminal domain contains the eEF-2 targeting domain. The calmodulin-binding region is located between amino acids 51 and 96. The amino acid sequence of the carboxyl-terminal domain of eEF-2 kinase displays similarity to several proteins, all of which contain repeats of a 36-amino acid motif that we named "motif 36".

Comparison of Folding Rates of Homologous Prokaryotic and Eukaryotic Proteins

The rate of polypeptide chain elongation is up to one order of magnitude faster in prokaryotic cells than in eukaryotes. Here we report that the rates of in vitro refolding of orthologous prokaryotic and eukaryotic proteins correlate with their differential rates of biosynthesis. The mitochondrial and cytosolic aspartate aminotransferases of chicken and aspartate aminotransferase of Escherichia coli show pairwise sequence identities of 41-48% and nearly identical three-dimensional structures. Nevertheless, the prokaryotic enzyme refolded 6 times faster (at 25 degrees C) than the eukaryotic isoenzymes after denaturation in 6 m guanidine hydrochloride. Prokaryotic malate dehydrogenase and lactate dehydrogenase also renatured faster than their orthologous eukaryotic counterparts, suggesting that evolutionary pressure has adapted the rate of folding to the rate of elongation of polypeptide chains.

Sporulation at minimum specific growth rate in Aspergillus nidulans chemostat culture predicted using protein synthesis efficiency estimations.

Ribosomal efficiency (RE) estimates provide a quantitative descriptor of intrinsic growth rate of cell populations using readily-obtainable experimental data. In Aspergillus nidulans chemostat cultures, RE increased linearly with growth rate over the range 25-60% of maximum growth rate (mu max), consistent with increasing ribosomal usage with increased growth rate. Above 60%, RE did not increase significantly, suggesting that all ribosomes were functional at 60% of mu max, further increases in growth rate, presumably resulting from increased polypeptide chain elongation rate. Extrapolating the linear part of the RE/growth rate curve predicted zero RE at a growth rate of 0.04 h-1. Chemostat steady state cultures at 0.04 h-1 contained spores (conidia), apparently undergoing a continuous sporulation/germination cycle. We propose that the RE estimates provide a means of predicting the value of minimum specific growth rate (mu min) below which net growth cannot take place.

The methyl group of the N6-methyl-N6-threonylcarbamoyladenosine in tRNA of Escherichia coli modestly improves the efficiency of the tRNA.

tRNA species that read codons starting with adenosine (A) contain N6-threonylcarbamoyladenosine (t6A) derivatives adjacent to and 3' of the anticodons from all organisms. In Escherichia coli there are 12 such tRNA species of which two (tRNA(Thr1)GGU and tRNA(Thr3)GGU) have the t6A derivative N6-methyl-N6-threonylcarbamoyladenosine (m6t6A37). We have isolated a mutant of E. coli that lacks the m6t6A37 in these two tRNA(Thr)GGU species. These tRNA species in the mutant are likely to have t6A37 instead of m6t6A37. We show that the methyl group of m6t6A37 originates from S-adenosyl-L-methionine and that the gene (tsaA) which most likely encodes tRNA(m6t6A37)methyltransferase is located at min 4.6 on the E. coli chromosomal map. The growth rate of the cell, the polypeptide chain elongation rate, and the selection of Thr-tRNA(Thr)GGU to the ribosomal A site programmed with either of the cognate codons ACC and ACU were the same for the tsaA1 mutant as for the congenic wild-type strain. The expression of the threonine operon is regulated by an attenuator which contains in its leader mRNA seven ACC codons that are read by these two m6t6A37-containing tRNA(Thr)GGU species. We show that the tsaA1 mutation resulted in a twofold derepression of this operon, suggesting that the lack of the methyl group of m6t6A37 in tRNA(Thr)GGU slightly reduces the efficiency of this tRNA to read cognate codon ACC.

Identification of the rrmA gene encoding the 23S rRNA m1G745 methyltransferase in Escherichia coli and characterization of an m1G745-deficient mutant.

An Escherichia coli mutant lacking the modified nucleotide m1G in rRNA has previously been isolated (G. R. Björk and L. A. Isaksson, J. Mol. Biol. 51:83-100, 1970). In this study, we localize the position of the m1G to nucleotide 745 in 23S rRNA and characterize a mutant deficient in this modification. This mutant shows a 40% decreased growth rate in rich media, a drastic reduction in loosely coupled ribosomes, a 20% decreased polypeptide chain elongation rate, and increased resistance to the ribosome binding antibiotic viomycin. The rrmA gene encoding 23S rRNA m1G745 methyltransferase was mapped to bp 1904000 on the E. coli chromosome and identified to be identical to the previously sequenced gene yebH.

Mutation in the structural gene for release factor 1 (RF-1) of Salmonella typhimurium inhibits cell division.

A temperature-sensitive mutant of Salmonella typhimurium LT2 was isolated. At the nonpermissive temperature cell division stopped and multinucleated filaments were formed. DNA, RNA, or protein synthesis was not affected until after about two generations. Different physiological conditions, such as anaerobiosis and different growth media, suppress the division deficiency at high temperatures. Certain mutations causing a reduced polypeptide chain elongation rate also suppress the division deficiency. The mutation is recessive and shown to be in the structural gene for release factor I (prfA). DNA sequencing of both the wild-type (prfA+) and mutant (prfA101) allele revealed a GC-to-AT transition in codon 168. Like other known prfA mutants, prfA101 can suppress amber mutations. The division defect in the prfA101 mutant strain could not be suppressed by overexpression of the ftsQAZ operon. Moreover, at the nonpermissive temperature the mutant shows a normal heat shock and SOS response and has a normal ppGpp level. We conclude that the prfA101-mediated defect in cell division is not directed through any of these metabolic pathways, which are all known to affect cell division. We speculate that the altered release factor I induces aberrant synthesis of an unidentified protein(s) involved in the elaborate process of septation.

1-Methylguanosine deficiency of tRNA influences cognate codon interaction and metabolism in Salmonella typhimurium

1-Methylguanosine (m1G) is present next to the 3' end of the anticodon (position 37) in tRNA(1,2,3,Leu), tRNA(1,2,3,Pro), and tRNA(3Arg). A mutant of Salmonella typhimurium lacks m1G in these seven tRNAs when grown at or above 37 degrees C, as a result of a mutation (trmD3) in the structural gene (trmD) for the tRNA(m1G37)methyltransferase. The m1G deficiency induced 24 and 26% reductions in the growth rate and polypeptide chain elongation rate, respectively, in morpholinepropanesulfonic acid (MOPS)-glucose minimal medium at 37 degrees C. The expression of the leuABCD operon is controlled by the rate with which tRNA(2Leu) and tRNA(3Leu) read four leucine codons in the leu-leader mRNA. Lack of m1G in these tRNAs did not influence the expression of this operon, suggesting that m1G did not influence the efficiency of tRNA(2,3Leu). Since the average step time of the m1G-deficient tRNAs was increased 3.3-fold, the results suggest that the impact of m1G in decoding cognate codons may be tRNA dependent. The trmD3 mutation rendered the cell more resistant or sensitive to several amino acid analogs. 3-Nitro-L-tyrosine (NT), to which the trmD3 mutant is sensitive, was shown to be transported by the tryptophan-specific permease, and mutations in this gene (mtr) render the cell resistant to NT. Since the trmD3 mutation did not affect the activity of the permease, some internal metabolic step(s), but not the uptake of the analog per se, is affected. We suggest that the trmD3-mediated NT sensitivity is by an abnormal translation of some mRNA(s) whose product(s) is involved in the metabolic reactions affected by the analog. Our results also suggest that tRNA modification may be a regulatory device for gene expression.

The methylthio group (ms2) of N6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A) present next to the anticodon contributes to the decoding efficiency of the tRNA.

A Salmonella typhimurium LT2 mutant which harbors a mutation (miaB2508::Tn10dCm) that results in a reduction in the activities of the amber suppressors supF30 (tRNA(CUATyr)), supD10 (tRNA(CUASer)), and supJ60 (tRNA(CUALeu)) was isolated. The mutant was deficient in the methylthio group (ms2) of N6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A), a modified nucleoside that is normally present next to the anticodon (position 37) in tRNAs that read codons that start with uridine. Consequently, the mutant had i6A37 instead of ms2io6A37 in its tRNA. Only small amounts of io6A37 was found. We suggest that the synthesis of ms2io6A occurs in the following order: A-37-->i6A37-->ms2i6A37-->ms2io6A37. The mutation miaB2508::Tn10dCm was 60% linked to the nag gene (min 15) and 40% linked to the fur gene and is located counterclockwise from both of these genes. The growth rates of the mutant in four growth media did not significantly deviate from those of a wild-type strain. The polypeptide chain elongation rate was also unaffected in the mutant. However, the miaB2508::Tn10dCm mutation rendered the cell more resistant or sensitive, compared with a wild-type cell, to several amino acid analogs, suggesting that this mutation influences the regulation of several amino acid biosynthetic operons. The efficiencies of the aforementioned amber suppressors were decreased to as low as 16%, depending on the suppressor and the codon context monitored, demonstrating that the ms2 group of ms2io6A contributes to the decoding efficiency of tRNA. However, the major impact of the ms2io6 modification in the decoding process comes from the io6 group alone or from the combination of the ms2 and io6 groups, not from the ms2 group alone.

Genetic Dissection of Synthesis and Function of Modified Nucleosides in Bacterial Transfer RNA

Publisher Summary This chapter discusses the transfer ribonucleic acid (tRNA) modification-deficient mutants that have been pivotal in unraveling their role in tRNA function. Unexpectedly, some modified nucleosides (m5U54, s4U8, and Q34), which occur frequently in tRNA, show no apparent functional impact on the tRNA based on the normal growth rate of the corresponding mutants. Other modified nucleosides (ms2ioGA37, mlG37, and W38, 39, 40) influence the function of the tRNA, and result in a reduced growth rate and polypeptide chain elongation rate for the mutant. Although, tRNA from all the three phylogenetic domains, archaea, bacteria, and eucarya, earlier called the kingdoms of archaebacteria, eubacteria, and eukaryotes, respectively contains modified nucleosides. A subset of these modified nucleosides is present in the tRNAs from all organisms. Moreover, some are even present in comparable positions in the tRNAs from all three domains. Therefore, the modified nucleosides may have been present in the tRNA of the progenitor. However, some modified nucleosides are domain-specific demonstrating that the presence of these modified nucleosides has evolved after the three phylogenetic domains had separated.

High Concentrations of ppGpp Decrease the RNA Chain Growth Rate: Implications for Protein Synthesis and Translational Fidelity During Amino Acid Starvation in Escherichia coli

We show that the RNA chain growth rate on lacZ is reduced by an elevated ppGpp level even in the absence of starvation. Under these conditions the polypeptide chain elongation rate is affected little, if at all. These results lead us to re-examine the role of ppGpp in the reduction of protein synthesis and translation fidelity during amino acid starvation. We find that ppGpp has little or no direct effect on translation rate or fidelity. Rather, the effects of ppGpp on translation are indirectly caused by the fact that ppGpp mRNA synthesis, making mRNA limiting for translation during amino acid starvation. The reduced level of mRNA thereby reduces the severity of the aminoacyl-tRNA limitation and, in turn, decreases mistranslation. Mistranslation in the starved relA strain therefore results from an increased severity of aminoacyl-tRNA limitation due to the failure of this strain to reduce mRNA levels by increasing the level of ppGpp. Finally, the initial rise of the ppGpp level in the starved stringent strain, followed by a characteristic reduction to a steady post-starved level, can now be explained by the initially high, and then decreasing number of "hungry" codons adjusted through the mRNA pool.

Regulation of macromolecular synthesis during nutritional shift-up in the fungusMucor

The dimorphic fungusMucor racemosus was grown anaerobically in the yeast form and shifted from a defined minimal medium (μ=0.17 doublings/h) to an enriched complex medium (μ=0.43 doublings/h). The measured rates of increase of several growth-related parameters displayed an immediate and complete adjustment to the higher growth rate without an intervening lag. Pulse-labeling experiments indicated that the rates of protein and RNA synthesis increased immediately and in parallel in response to the shift. The rate of polypeptide chain elongation immediately increased by approximately 60% to support the new higher rate of protein accumulation, but later declined to near pre-shift values. The accelerated rate of protein accretion was apparently sustained by a gradual increase in the total number of ribosomes per weight of cellular material and by a more rapid increase in the percentage of ribosomes recruited into polysomes at a given time. Thus, this simple eukaryote displays a much more rapid and varied mechanism of response to nutritional shift-up conditions than is classically observed in bacteria.

Altered growth-rate-dependent regulation of 6-phosphogluconate dehydrogenase level in hisT mutants of Salmonella typhimurium and Escherichia coli

In Escherichia coli, the level of 6-phosphogluconate dehydrogenase is directly proportional to the cellular growth rate during growth in minimal media. This contrasts with the report by Winkler et al. (M. E. Winkler, J. R. Roth, and P. E. Hartman, J. Bacteriol. 133:830-843, 1978) that the level of the enzyme in Salmonella typhimurium LT-2 strain SB3436 is invariant. The basis for the difference in the growth-rate-dependent regulation between the two genera was investigated. Expression of gnd, which encodes 6-phosphogluconate dehydrogenase, was growth rate uninducible in strain SB3436, as reported previously, but it was 1.4-fold growth rate inducible in other S. typhimurium LT-2 strains, e.g., SA535. Both the SB3436 and SA535 gnd genes were growth rate inducible in E. coli K-12. Moreover, the nucleotide sequences of the regulatory regions of the two S. typhimurium genes were identical. We concluded that a mutation unlinked to gnd is responsible for the altered growth rate inducibility of 6-phosphogluconate dehydrogenase in strain SB3436. Transductional analysis showed that the altered regulation is due to the presence of a mutation in hisT, the gene for the tRNA modification enzyme pseudouridine synthetase I. A complementation test showed that the regulatory defect conferred by the hisT mutation was recessive. In E. coli, hisT mutations reduced the extent of growth rate induction by the same factor as in S. typhimurium. The altered regulation conferred by hisT mutations was not simply due to their general effect of reducing the polypeptide chain elongation rate, because miaA mutants, which lack another tRNA modification and have a similarity reduced chain growth rate, had higher rather than lower 6-phosphogluconate dehydrogenase levels. Studies with genetic fusions suggested that hisT mutations lower the gnd mRNA level. The data also indicated that hisT is involved in translational control of gnd expression, but not the aspect mediated by the internal complementary sequence.

Анализ кинетической схемы стадии элонгации белкового синтеза в рамках гипотезы о стереоспецифической стабилизации кодон-антикодоновых комплексов на рибосоме. 1. Скорость элонгации полипептидных цепей

Анализ кинетической схемы стадии элонгации белкового синтеза в рамках гипотезы о стереоспецифической стабилизации кодон-антикодоновых комплексов на рибосоме. 1. Скорость элонгации полипептидных цепей

Regulation of polypeptide chain initiation and activity of initiation factor eIF-2 in Chinese-hamster-ovary cell mutants containing temperature-sensitive aminoacyl-tRNA synthetases.

The regulation of polypeptide chain initiation has been investigated in extracts from a number of well-characterized Chinese hamster ovary (CHO) cell mutants containing different temperature-sensitive aminoacyl-tRNA synthetases. These cells exhibit a large decline in the rate of initiation when cultures are shifted from the permissive temperature of 34 degrees C to the non-permissive temperature of 39.5 degrees C. During a brief incubation with [35S]Met-tRNAMetf or [35S]methionine, formation of initiation complexes on native 40S ribosomal subunits and 80S ribosomes is severely impaired in extracts from the mutant cell lines exposed to 39.5 degrees C. Wild-type cells exposed to 39.5 degrees C do not show any inhibition of protein synthesis or initiation complex formation. Inhibition of formation of 40S initiation complexes in the extracts from mutant cells, incubated at the non-permissive temperature, is shown to be independent of possible changes in mRNA binding or the rate of polypeptide chain elongation and is not due to any decrease in the total amount of initiation factor eIF-2 present. However, assays of eIF-2 X GTP X Met-tRNAMetf ternary complex formation in postribosomal supernatants from the temperature-sensitive mutants reveal a marked defect in the activity of eIF-2 after exposure of the cells to 39.5 degrees C and addition of exogenous eIF-2 to cell-free protein-synthesizing systems from cells incubated at 34 degrees C and 39.5 degrees C eliminates the difference in activity between them. The activity of the initiation factor itself is not directly temperature-sensitive in the mutant CHO cells. The results suggest that the activity of aminoacyl-tRNA synthetases can affect the ability of eIF-2 to bind Met-tRNAMetf and form 40S initiation complexes in intact cells, indicating a regulatory link between polypeptide chain elongation and chain initiation.

Increased rates of polypeptide chain elongation in placental explants from human diabetics.

Average rates of polypeptide chain elongation were determined in placental explants of first trimester and term placentas from both normal and diabetic human pregnancies. Average ribosome half-transit times were determined by measuring the kinetics of transfer of labeled polypeptides from polysomal-bound to released polypeptides. The average half-transit time decreases from 75 sec per ribosome in first trimester explants to 56 sec per ribosome in term placentas. The average polypeptide molecular weights synthesized by explants from first trimester and in term are 49,300 and 49,600, respectively, which are not significantly different. The average elongation rates for first trimester and term placental explants are 172 and 231 amino acids per minute per ribosome, respectively, which are significantly different. Moreover, the average polypeptide molecular weight synthesized by term placentas from diabetic pregnancies is 48,200, while the average ribosome half-transit time is 40 sec. Thus, ribosomes from explants of term placenta from diabetics move along the average message at a much higher speed than do ribosomes in normal term tissue. The assembly rate of amino acid into polypeptide in explant of placenta of diabetic mothers is 314 amino acids per minute, which is significantly faster than 231 amino acids per minute in normal term tissue. These findings indicate that during placental development and in diabetic pregnancy there is a large change in the actual rates at which amino acids are added to the nascent polypeptide chain--i.e., the rates in polypeptide chain elongation. Therefore, translation-level regulation of protein synthesis in placenta plays a significant part in the magnitude of the response to developmental and other physiological stimulations.

Coordinate regulation of polypeptide chain initiation and elongation in rabbit reticulocytes.

Under normal conditions, reticulocytes synthesize alpha- and beta-globin polypeptides at equal rates. Incubation in the absence of hemin or under anoxia or hypertonic stress (100 mM excess NaCl) reduces the rate of protein synthesis to 30-50% of control levels. However, only hemin deprivation causes a reduction in polyribosome size and preferential inhibition of alpha-globin synthesis consistent with specific reduction in the rate of polypeptide chain initiation. Polyribosomal profiles are unaffected by anoxic or hypertonic stress and the ratio of alpha:beta globin synthesis remains close to unity. Measurement of ribosome transit time indicates that anoxic or hypertonic stress causes a decrease in the rate of polypeptide chain elongation that varies with the degree of inhibition of protein synthesis. Ribosomes isolated from stressed cells exhibit a reduced ability to bind 35S-met-tRNAf, suggesting that the ability to form initiation complexes is also impaired. These results suggest that reticulocytes, unlike nucleated cell lines, can coordinately reduce rates of initiation and elongation in response to certain physiological stresses.

Transacylation rates of (aminoacyl)adenosine moiety at the 3'-terminus of aminoacyl transfer ribonucleic acid.

The rates of migration of the aminoacyl group (transacylation) between 2'-O-(aminoacyl)-tRNA and 3'-O-(aminoacyl)-tRNA were studied by the nuclear magnetic resonance (NMR) analyses of 3'-terminal fragment models, with regard to the significance of transacylation in the process of protein biosynthesis. 2'(3')-O-L-Alanyladenosine, -valyladenosine, -isoleucyladenosine, -phenylalanyladenosine, and -methionyladenosine, and 2'(3')-O-L-phenylalanyladenosine 5'-phosphate and methionyladenosine 5'-phosphate were chemically synthesized, and the rates of transacylation in deuterated buffer were directly measured by the NMR saturation transfer method. The dependences of transacylation rates on p2H and temperature were analyzed. The results indicate that the transacylation rates are significantly affected by the ionization states of the alpha-amino group of the amino acid moiety but not by the presence of the 5'-phosphate group of the adenylate moiety. The second-order rate constants for the base-catalyzed transacylation reactions were also determined for the ionized form (with alpha-N2H3+ group) of (aminoacyl)adenosines. The transacylation rates of (aminoacyl)adenosines in 1H2O solution at p1H 7.3 and 37 degrees C (intracellular environment) were evaluated as 3-11 s-1 for the 2' leads to 3' transacylation and 1-4 s-1 for the 3' leads to 2' transacylation, indicating that the transacylation rate of free aminoacyl-tRNA is slower than the overall rate of polypeptide chain elongation per ribosome. This suggests the presence of some enzymatic factor for enhancing the transacylation rates of aminoacyl-tRNAs in the polypeptide chain elongation process in vivo.