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Biomedical subjects

A R Subramanian

Publications and source records attributed to A R Subramanian.

At least 91 records · Page 5Linked to original sources
PubMed1979

Characterization of a mutant form of ribosomal protein S1 from Escherichia coli.

An altered form of ribosomal protein S1 from a mutant of Escherichia coli has been isolated and characterized. The mutant protein (denoted m1-S1) has a molecular weight of 57,000 as shown by sodium dodecyl sulfate-gel electrophoresis and the same NH2-terminal sequence as wild type S1. Protein m1-S1 binds poly(U) in the same manner as protein S1 and is active in protein synthesis with either synthetic or natural mRNA. Thus, about 75% of the sequence of protein S1 (which includes the NH2-terminal region) contains essentially all the functional domains of this protein involved in protein biosynthesis.

Cross Reactions↗
PubMed1979

Protein S1 from Escherichia coli ribosomes: an improved isolation procedure and shape determination by small-angle X-ray scattering.

Ribosomal protein S1 from Escherichia coli was studied in solution by small-angle X-ray scattering and the following parameters were obtained. The radius of gyration R = 8.0 +/- 0.2 nm; largest diameter D = 28 nm; molecular weight = (8--9) x 10(4). The data also yielded (with the assumption of a rigid particle with almost constant electron density) two radii of gyration of cross-section Rq1 = 2.5 +/- 0.1 nm and Rq2 = 1.05 +/- 0.05 nm and molecular volume = 140 nm3. The experimental scattering curve of S1 was compared with the theoretical scattering curves for several rigid triaxial homogeneous bodies and the closest fit was given by that of a flat elliptical cylinder with the dimensions of 4.5 nm and 0.88 nm for the two semiaxes and 26.5 nm for height. The results from the present X-ray scattering studies and those from limited proteolytic digestion of protein S1 [J. Mol. Biol. 127, 41--54, (1979)] support the notion that the structure of protein S1 is organized into two distinct subdomains within its elongated overall shape. Protein S1 was purified for this study by an efficient procedure which yielded 12 mg S1/g ribosomes. The isolated protein was fully active in functional tests both before and after X-ray irradiation.

Bacterial Proteins↗
PubMed1979

Nucleic acid binding and unfolding properties of ribosomal protein S1 and the derivatives S1-F1 and m1-S1.

The nucleic acid binding and unwinding properties of wild-type Escherichia coli ribosomal protein S1 have been compared to those of a mutant form and a large trypsin-resistant fragment, both reported recently [J. Mol. Biol. 127, 41-45 (1979) and J. Biol. Chem. 254, 4309-4312 (1979). The mutant (m1-S1) contains 77% and the fragment (S1-F1) 66% of the polypeptide chain length (approximately 600 amino acid residues) of protein S1. The mutant is active in protein synthesis in vitro; the fragment, although retaining one or more of the functional domains of S1, is inactive in protein synthesis. We find that m1-S1 is is almost as effective as S1 in binding to poly(rU), phage MS2 RNA and simian virus 40 (SV40) DNA, and in unfolding poly(rU) and the helical structures present in MS2 RNA and phi X174 viral DNA. S1-F1, however, binds to poly(rU) and denatured SV40 DNA, but not to MS2 RNA. It unfolds neither poly(rU), nor the residual secondary structure of MS2 RNA or phi X174 viral DNA. Thus, there appears to be a correlation between the loss in ability of S1 to unwind RNA and the loss in its ability to function in protein synthesis.

Bacterial Proteins↗
PubMed1978

Two novel growth cycle-reflecting proteins associated with Escherichia coli ribosomes.

The isolation and characterization of two low molecular weight, growth cycle-reflecting proteins which are associated with Escherichia coli ribosomes is described. These proteins which show greatly enhanced stoichiometry in ribosomes derived from post-exponential cells, exhibit molecular weights of 16 600, and 11 700 on sodium dodecyl sulfate (SDS) gel electrophoresis. Both proteins are highly acidic, the larger being the most acidic protein associated with the ribosome. Their amino acid compositions are unique and distinguish them from all other ribosomal proteins. Neither of the proteins showed crossreaction with either anti-L7 or anti-S6 sera although their electrophoretic behavior resembles that of L7 and S6. During the purification we have also isolated small amounts of three low molecular weight proteins showing some immunological homology with L7/L12. The isolated proteins were found to be without effect on the in vitro translation of f2-RNA, indicating that these adaptive modifications of the ribosome do not seriously affect its intrinsic activity.

Amino Acids↗
PubMed1978

Specific association of two homologous DNA-binding proteins to the native 30-S ribosomal subunits of Escherichia coli.

The native 30-S ribosomal subunits from Escherichia coli are shown to be associated with two proteins which are different from the known ribosome-associated and ribosomal proteins. Neither protein is foune on native 50-S subunits or on intact ribosomes in the cell extract. The purified proteins re-bind in vitro to free 30-S subunits, but do not bind to either free 50-S subunits or intact ribosomes. The proteins, denoted NS1 and NS2, have been purified and characterized. Both proteins showed the same molecular weight of 9500 by sodium dodecyl sulfate gel electrophoresis but 34 000 by gel filtration. Upon treatment with cross-linking reagents the purified proteins gave higher molecular weight species up to the tetrameric ones showing that they exist in solution as tetramers. The amino acid compositions, tryptic fingerprint patterns and N-terminal sequences of the two proteins have been determined. These data show that NS1 and NS2 possess distinct primary structures but with extensive sequence homology. Antibodies raised against the purified proteins cross-reacted in double immuno-diffusion tests confirming further the homology. Because of the similarity in properties a sample of the DNA-binding protein HD (Berthold, V. and Geider, K. (1976) Eur. J. Biochem. 71, 443--449) was compared to NS1 and NS2. In terms of several criteria, the protein HD is found to be a mixture of two proteins, namely NS1 and NS2. The present report is the first instance of an association of DNA-binding proteins to the ribosome.

Amino Acid Sequence↗
PubMed1978

Variations in the protein pattern of Ehrlich ascites tumor cell polyribosomes.

The two-dimensional gel electrophoretic protein pattern of polyribosomes from Ehrlich ascites tumor cells which have been fasted in a nutritionally deprived medium was compared to the corresponding pattern derived from unstarved cells. The proteins were labeled with 3H-labeled amino acids and their counts determined to make the comparison quantitative. Three proteins were found in the "fasted" polyribosomes in 4--6-fold greater amounts than in the fed control.

Amino Acids↗
PubMed1978

Metabolic stability of the two forms of initiation factor IF-3 in Escherichia coli during the growth cycle.

Possible alteration in the ratio of the long and short forms of initiation factor IF-3 (FEBS Lett. 79, 264-275, 1977) during the growth cycle of Escherichia coli was examined. The ratio was found to remain unchanged between the exponential and stationary growth phases. Contrary to an earlier report (Eur. J. Biochem. 29, 319-325, 1972), the total amount of IF-3 relative to the ribosome content in stationary phase cells was essentially the same as in midlogarithmic phase cells. The activity of IF-3, assayed after its separation from other initiation factors by chromatography, was also the same in extracts from midlogarithmic and stationary phase cells. The data show that in Escherichia coli the ratio of IF-3/ribosome is maintained constant. The ribosomes themselves have been shown to retain virtually full activity in vitro during this transition indicating that growth-cycle-dependent biochemical modifications of the ribosome do not affect its protein synthetic capacity per se.

Cell Division↗
PubMed1977

Exchange of individual ribosomal proteins between ribosomes as studied by heavy isotope-transfer experiments.

Whether the individual ribosomal proteins undergo exchange between robosomes in vivo during cell growth was examined using heavy isotope transfer methodology. E. coli was grown first in a heavy isotope medium in the presence of [3H] leucine and then transferred to normal medium and allowed to grow for one generation in the presence of [14C] leucine. The "heavy" and "light" ribosomes that were present in such cells were separated by sedimentation and the ribosomal proteins resolved by two-dimensional gel electrophoresis. The individual proteins were burnt in O2 and their contents of [3H] and [14C] labels determined. From the analysis of the data we find that the great majority of the ribosomal proteins of E. coli does not undergo exchange during cell growth. Proteins which were found to exchange to varying levels in different transfer experiments were S1, S2, L7/L12, L9, L10 and L33. All of them except L9 exchanged to the same levels in control experiments in which separately grown heavy and light cells were mixed and processed. These proteins therefore undergo exchange during cell breakage and ribosome isolation. Protein L9 consistently showed appreciably greater exchange in transfer experiments as compared to the controls suggesting that it may exchange in vivo.

Bacterial Proteins↗
PubMed1976

Isolation and characterization of a growth-cycle-reflecting, high-molecular-weight protein associated with Escherichia coli ribosomes.

A high-molecular-weight, acidic protein, acidic protein whose amount per ribosomal particle depends on the growth cycle is shown to be associated with Escherichia coli ribosomes. The protein is associated with ribosomes from cells harvested at different phases of the growth cycle, but a large increase in its amount is seen with ribosomes from post-exponential phase cells. The protein is only slightly washed off the ribosomes by 1 M NH4CL. When ribosomes are dissociated it remains entirely with the 30-S subunits. We have purified the protein to homogeneity. It has a molecular weight of 70 000 and its amino acid composition showed some resemblance to that of ribosomal protein S1. However, the two proteins ar-shown variation in the acetylation of L12 during the growth cycle, indicated that certain proteins of (or associated with) E. coli ribosmes may carry specific biochemical roles connected with cellular adaption toward the stationary phase.

Amino Acids↗