The Effect of Various Nucleotides on the Helical Nature of a Ribosomal Protein(s) from Escherichia coli

Abstract

In the presence of GTP or GDP, there is a decrease in the circular dichroic absorption of ribosomal proteins L7 and L12 at 221-222 nm, suggesting that these nucleotides influence the helical content of these proteins.All of the proteins from the 30S and 50S ribosomal subunits of Escherichia coli have been purified to homogeneity (1-5), although little is known about the function of these proteins in polypeptide synthesis. Recently Hamel et al. (6) and our laboratory (7) reported the isolation of a ribosomal protein fraction from E. coli that is required for the function of both elongation factors Tu (EF-Tu) and G (EF-G). We concluded-on the basis of disc gel electrophoresis at pH 4.5, amino-acid analysis, and molecular weight-that the purified material contains two proteins identical to the Al and A2 proteins described by M6ller et at. (8), and proteins L7 and L,2 isolated by Wittmann and his coworkers (9-11). The latter designations will be used in this report. These two proteins appear identical by all physical parameters measured. Thus, they are both acidic, have molecular weights of about 13,000, have a high a-helical content, similar amino-acid analysis, and are immunologically crossreactive. The only difference appears to be that L7 contains an N-acetylated amino-terminal serine, whereas in L12 the N-terminal serine is unblocked (12).In view of the high helicity of proteins L7 and L12, and the fact that their presence on the ribosome is required for the GTP-dependent EF-Tu and EF-G reactions in protein synthesis, it seemed of interest to investigate whether the helical nature of these proteins could be altered in any way. For example, one could speculate that these helical proteins, by virtue of their ability to undergo a reversible conformational change, may be involved in movement of the ribosome along messenger RNA. The present report describes the effect of various nucleotides on the circular dichroism (CD) spectra of a mixture of proteins L7 and L12. MATERIALS AND METHODSE. coli ribosomes were isolated and washed in a buffered 1 M NH4C1 solution (13). These salt-washed ribosomes were treated with 40% ethanol in the presence of 1 M NH4Cl (6, 7), and the ribosomal proteins L7 and L12 were purified together (7) from the ethanolic supernatant. The CD spectra were performed at room temperature in a 0.01-cm cell on a Cary 61 recording spectropolarimeter. The standard mixture contained 10 mM (each) of Tris HCl buffer (pH 7.4), MgC12, NH4C1, and, where indicated, the following were added (final concentrations): 0.7 mg/ml of a mixture of ribosomal proteins L7 and L12, 0.7 mg/ml of EF-G, and 2.5 mM or 4 mM nucleotide. The data are expressed as the mean residue ellipticity (0) (deg. cm2/dmol), and are not corrected for the refractive index of the solvent. A value of 117 was used as the mean residue weight of the ribosomal proteins, and 127 for the mean residue weight of EF-G.RESULTS AND DISCUSSION Fig. 1 shows the CD spectra of the ribosomal proteins and of EF-G. The spectrum of the mixture of L7 and L12...

Effect of propan-2-ol on enzymic and structural properties of elongation factor G

“…However, [0]285 increased considerably at increasing concentrations of the alcohol; the enhancement observed in the presence of 200% (v/v) propan-2-ol was not reproduced by 200% ethanol (results not shown) which was previously found to be a minor effector of GTPaseP (De Vendittis et al, 1986). It is worth mentioning that the value of -9500 degrees cm2 dmol-1 determined for [1]22o was similar to that previously reported (Brot et al, 1972;Alakhov et al, 1979).…”

Oxidation of the methionine residues of Escherichia coli ribosomal protein L12 decreases the protein's biological activity.

“…The enzyme that catalyzes the acetylation has been purified, and it has been shown that free L12 is acetylated 10 times faster than ribosomal-bound L12 (16). It is also of interest that these proteins lack cysteine, histidine, tryptophan, and tyrosine, are very acidic (pI = 4.7-4.9), and have a high degree of secondary structure with >45% helical content (17)(18)(19). The proteins in solution form dimers (14,20,21) that may be the functional form on the ribosome and also bind tightly to ribosomal protein L10 (22,23).…”