scholarly journals Defects in the assembly of ribosomes selected for β-amino acid incorporation

2019 ◽  
Author(s):  
Fred R. Ward ◽  
Zoe L. Watson ◽  
Omer Ad ◽  
Alanna Schepartz ◽  
Jamie H. D. Cate
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ribosome Assembly ◽  
Amino Acid Incorporation ◽  
Ribosome Engineering ◽  
Acid Incorporation ◽  
Weak Activity ◽  
Peptidyltransferase Center

AbstractRibosome engineering has emerged as a promising field in synthetic biology, particularly concerning the production of new sequence-defined polymers. Mutant ribosomes have been developed that improve the incorporation of several non-standard monomers including D-amino acids, dipeptides, and β-amino acids into polypeptide chains. However, there remains little mechanistic understanding of how these ribosomes catalyze incorporation of these new substrates. Here we probed the properties of a mutant ribosome–P7A7–evolved for better in vivo β-amino acid incorporation through in vitro biochemistry and cryo-electron microscopy. Although P7A7 is a functional ribosome in vivo, it is inactive in vitro, and assembles poorly into 70S complexes. Structural characterization revealed large regions of disorder in the peptidyltransferase center and nearby features, suggesting a defect in assembly. Comparison of RNA helix and ribosomal protein occupancy with other assembly intermediates revealed that P7A7 is stalled at a late stage in ribosome assembly, explaining its weak activity. These results highlight the importance of ensuring efficient ribosome assembly during ribosome engineering towards new catalytic abilities.

Effect of Cycloheximide on In Vitro and In Vivo Protein Synthesis by Certain Tissues of Rats and Pigeons with Special Reference to Muscle

1973 ◽  
Vol 51 (12) ◽  
pp. 933-941 ◽  
Author(s):  
Njanoor Narayanan ◽  
Jacob Eapen
Keyword(s):  
Amino Acids ◽  
Body Weight ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Amino Acid Incorporation ◽  
Contrast Administration ◽  
Acid Incorporation ◽  
Tissue Homogenates

The effect of cycloheximide in vitro and in vivo on the incorporation of labelled amino acids into protein by muscles, liver, kidneys, and brain of rats and pigeons was studied. In vitro incorporation of amino acids into protein by muscle microsomes, myofibrils, and myofibrillar ribosomes was not affected by cycloheximide. In contrast, administration of the antibiotic into intact animals at a concentration of 1 mg/kg body weight resulted in considerable inhibition of amino acid incorporation into protein by muscles, liver, kidneys, and brain. This inhibition was observed in all the subcellular fractions of these tissues during a period of 10–40 min after the administration of the precursor. Tissue homogenates derived from in vivo cycloheximide-treated animals did not show significant alteration in in vitro amino acid incorporation with the exception of brain, which showed a small but significant enhancement.

Incorporation of C14-amino acids into protein of isolated diaphragms: effect of epinephrine and norepinephrine

1960 ◽  
Vol 198 (1) ◽  
pp. 54-56 ◽  
Author(s):  
Ira G. Wool
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Glucose Concentration ◽  
Muscle Protein ◽  
Effective Concentration ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Minimal Effective Concentration

When diaphragms isolated from normal rats were incubated with a C14-amino acid the addition of epinephrine or norepinephrine decreased incorporation of C14 into muscle protein. The inhibition occurred whether epinephrine was added in vitro or administered in vivo. The minimal effective concentration of epinephrine in vitro was 0.1 µg/ml. When the glucose concentration in the medium was raised to 300 mg % or more the epinephrine induced inhibition of amino acid incorporation into muscle protein was no longer observed.

scholarly journals FRUCTOSE-AMINO ACIDS IN LIVER: STIMULI OF AMINO ACID INCORPORATION IN VITRO

1955 ◽  
Vol 215 (1) ◽  
pp. 111-124 ◽  
Author(s):  
Henry Borsook ◽  
Adolph Abrams ◽  
Peter H. Lowy
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Incorporation ◽  
Acid Incorporation

Reduced and S-carboxymethylated human growth hormone: a probe for diabetogenic action

1984 ◽  
Vol 247 (5) ◽  
pp. E639-E644
Author(s):  
C. M. Cameron ◽  
J. L. Kostyo ◽  
J. A. Rillema ◽  
S. E. Gennick
Keyword(s):  
Growth Hormone ◽  
Amino Acid ◽  
Human Growth Hormone ◽  
Human Growth ◽  
Mouse Mammary Gland ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Hypophysectomized Rats

The biological activity profile of reduced and S-carboxymethylated human growth hormone (RCM-hGH) was determined to establish its suitability for study of the diabetogenic property of hGH. RCM-hGH was found to have greatly attenuated in vivo growth-promoting activity in the 9-day weight-gain test in hypophysectomized rats (approximately 1%) and to have a similar low order of in vitro activity in stimulating amino acid incorporation into the protein of the isolated rat diaphragm. RCM-hGH also only had approximately 1% of the in vitro insulin-like activity of the native hormone on isolated adipose tissue from hypophysectomized rats. In contrast, RCM-hGH retained substantial in vivo diabetogenic activity in the ob/ob mouse, appearing to have approximately 50% of the activity of the native hormone. RCM-hGH was also found to retain significant, although attenuated (25%), in vitro lactogenic activity when tested for the ability to stimulate amino acid incorporation into a casein-rich protein fraction in mouse mammary gland explants. Because RCM-hGH exhibits a high degree of diabetogenic activity, although lacking significant anabolic or insulin-like activities, it will be useful as a "monovalent" probe for the study of the molecular mechanism of the diabetogenic action of GH.

scholarly journals Action of growth hormone in vitro on the net uptake and incorporation into protein of amino acids in muscle from intact rabbits given protein-deficient diets

1976 ◽  
Vol 35 (1) ◽  
pp. 1-10 ◽  
Author(s):  
M. R. Turner ◽  
P. J. Reeds ◽  
K. A. Munday
Keyword(s):  
Amino Acids ◽  
Growth Hormone ◽  
Protein Synthesis ◽  
Amino Acid ◽  
De Novo ◽  
Amino Acid Uptake ◽  
Acid Uptake ◽  
Amino Acid Incorporation ◽  
Acid Incorporation

1. Net amino acid uptake, and incorporation into protein have been measured in vitro in the presence and absence of porcine growth hormone (GH) in muscle from intact rabbits fed for 5 d on low-protein (LP), protein-free (PF) or control diets.2. In muscle from control and LP animals GH had no effect on the net amino acid uptake but stimulated amino acid incorporation into protein, although this response was less in LP animals than in control animals.3. In muscle from PF animals, GH stimulated both amino acid incorporation into protein and the net amino acid uptake, a type of response which also occurs in hypophysectomized animals. The magnitude of the effect of GH on the incorporation of amino acids into protein was reduced in muscle from PF animals.4. The effect of GH on the net amino acid uptake in PF animals was completely blocked by cycloheximide; the uptake effect of GH in these animals was dependent therefore on de novo protein synthesis.5. It is proposed that in the adult the role of growth hormone in protein metabolism is to sustain cellular protein synthesis when there is a decrease in the level of substrate amino acids, similar to that which occurs during a short-term fast or when the dietary protein intake is inadequate.

scholarly journals Biotin-mediated protein biosynthesis

1974 ◽  
Vol 140 (3) ◽  
pp. 549-556 ◽  
Author(s):  
R. L. Boeckx ◽  
K. Dakshinamurti
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Rat Liver ◽  
Intestinal Mucosa ◽  
Protein Biosynthesis ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Stimulation Of

The effect of administration of biotin to biotin-deficient rats on protein biosynthesis was studied. Biotin treatment resulted in stimulation by more than twofold of amino acid incorporation into protein, both in vivo and in vitro in rat liver, pancreas, intestinal mucosa and skin. Analysis of the products of amino acid incorporation into liver proteins in vivo and in vitro indicated that the synthesis of some proteins was stimulated more than twofold, but others were not stimulated at all. This indicates a specificity in the stimulation of protein synthesis mediated by biotin.

THYROXINE STIMULATION OF AMINO ACID INCORPORATION INTO MITOCHONDRIAL PROTEIN: DIFFERENCES BETWEEN IN VIVO AND IN VITRO EFFECTS

1973 ◽  
Vol 72 (4) ◽  
pp. 684-696 ◽  
Author(s):  
Amirav Gordon ◽  
Martin I. Surks ◽  
Jack H. Oppenheimer
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Mitochondrial Protein ◽  
Leucine Incorporation ◽  
Mitochondrial Protein Synthesis ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Stimulation Of

ABSTRACT The in vivo and in vitro stimulation of rat hepatic mitochondrial protein synthesis by thyroxine (T4) was compared. In confirmation of Buchanan & Tapley (1966). T4 added to isolated mitochondria rapidly stimulated [14C] leucine incorporation into mitochondrial protein. The in vitro stimulation was reversed after T4 was removed by incubating the mitochondria with bovine serum albumin (BSA). The decrease in T4 stimulation of protein synthesis appeared proportional to the T4 removed by BSA. Thus, it appears probable that exchangeable T4 controls the in vitro system. In contrast, the increase in mitochondrial protein synthesis which was observed 3 to 4 days after pretreatment of hypothyroid rats with labelled and non-radioactive T4 was not reversed by BSA treatment. Moreover, mitochondrial radioactivity could not be extracted with albumin. The in vivo phenomenon does not, therefore, appear to be related to exchangeable hormone in the mitochondria. Furthermore, the estimated quantity of T4 associated with mitochondria after in vivo stimulation was at least two orders of magnitude less than that required to produce comparable stimulation of mitochondrial protein synthesis in vitro. These findings strongly suggest that in vitro and in vivo stimulation of amino acid incorporation by T4 may be mediated by different biochemical mechanisms.

EFFECTS OF DIHYDROSTREPTOMYCIN ON AMINO ACID INCORPORATION INTO THE PROTEINS OF M. TUBERCULOSIS (BCG)

1959 ◽  
Vol 37 (1) ◽  
pp. 687-697
Author(s):  
E. Stachiewicz ◽  
J. H. Quastel
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Sodium Stearate ◽  
Ehrlich Ascites Carcinoma ◽  
Inhibitory Effect ◽  
Total Radioactivity ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Drug Concentrations

A study has been made of the effects of dihydrostreptomycin on amino acid incorporation into the proteins of M. tuberculosis (BCG). Suspensions of this organism on incubation at 37° with glycine-1-C14give rise, aerobically, to labelled proteins in which 80% of the radioactivity appears in the glycine and serine moieties of the proteins and about 20% in alanine and aspartic acid. In presence of glycine-2-C14, radioactivity appears in a larger number of amino acids of the protein. Incubation with serine-3-C14leads to a distribution of radioactivity in the amino acids in BCG proteins but alanine-1-C14and valine-1-C14give rise to proteins with the radioactivity almost entirely in the corresponding amino acids. The process of aerobic incorporation of radioactivity from glycine-1-C14in BCG proteins is stimulated by the presence of glucose, glycerol, sodium pyruvate, sodium stearate, or sodium benzoate in the medium in which the cells are incubated, the rate of incorporation being approximately constant over a period of 4 hours. The incorporation depends largely on the presence of oxygen. Dihydrostreptomycin (33 μg per ml) markedly inhibits labelling of proteins in the cell suspensions in presence of radioactive amino acids, the inhibition increasing with concentration of the streptomycin to an optimal concentration of 200 μg/ml. Penicillin and isonicotinic hydrazide are inactive but chloromycetin is an effective inhibitor. Cyanide, arsenite, and azide are inhibitory. The presence of lecithin stimulates incorporation of radioactivity from glycine-1-C14into BCG proteins. Dihydrostreptomycin inhibitions of amino acid incorporation into BCG proteins increase with time of incubation of the cells with the drug. Concentrations of dihydrostreptomycin that inhibit labelled amino acid incorporation into labelled proteins by 50% have no effect on BCG respiration. The drug has no inhibitory effect on labelled amino acid incorporation in E. coli or Ehrlich ascites carcinoma cells in vitro but is effective with M. phlei. It does not affect selectively the distribution of radioactivities of the component amino acids of BCG proteins; only the total radioactivity incorporated into the proteins is diminished. The results lead to the conclusion that dihydrostreptomycin brings about an inhibition of protein synthesis in the BCG strain of M. tuberculosis at concentrations at which it exerts antibiotic effects.

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