50-S Ribsomal Proteins. Peptide Studies on Two Acidic Proteins, A1 and A2, Isolated from 50-S Ribosomes of Escherichia coli

Abstract

Peptide studies on two acidic proteins, A, and A,, from 50-S ribosomes of Escherichia coli I. Amino acid compositions of the tryptic peptides of the two proteins are identical, with 2. The N-terminal amino acid sequence of A,-protein is Ser-Ile-Thr-Lys, while that of 3. The estimated number of 120 amino acid residues in each polypeptide chain is consistent 4. I n 500/, of the polypeptide chains, in both A,-or A,-protein, it specific lysine residue is indicate a closely related primary structure. exception of the amino terminal peptide.A,-protein is N-acetyl-Ser-Ile-Thr-Lys.with the physical molecular weight estimate.replaced by s-N-monomethyl-lysine.As reported in the preceding paper and their biological activity [S] which seems to be related to the phosphate energy liberation in the translocase step.The main objective of this study has been the precise determination of the difference in chemical structure between the two proteins. Thorough analysis of the tryptic peptides of the protein revealed that both have probably an identical primary structure and that the difference between the two is only confined to an acetyl group on the amino terminal end of the A,-protein, which is absent in A,-protein.From the peptide compositions the chemical molecular weight of the A-proteins have been estimated. MATERIALS AND METHODS Preparation of Tryptic PeptidesThe purification of A-protein, isolation and amino acid composition of A,-and A,-protein have been described elsewhere [i].Unusual Abbreviation. Dansyl, 1-dimethyl-aminonaphthalcne-5-sulphonyl.2 mg protein, determined by the microbiuret assay [l], was dissolved in 1 ml lo/, (NH,),CO, (pH 8.85) to which 20 p1 of a I mg/ml solution of trypsin treated with ~-l-tosyl-amido-2-phenylethylchloromethylketone, (Serva, Heidelberg, Germany) in 0.001 N HC1 was added to start the reaction at 37 "C. A second aliquot of enzyme (20 pg) was added after 1 h, the total digestion time being 4 h, after which the digest was freeze-dried, dissolved in 1 .O ml water and freeze-dried again. This residue was used directly for peptide mapping. Tryptic peptides for column chromatography were prepared by a 6-h digestion in dilute ammonia on a Radiometer pH-stat a t p H 8.0,37 "C. The enzyme to substrate weight ratio was also 1 to 50. The mixture was then frozen and lyophilized once. Separation of the Tryptic PeptidesPeptide maps were made essentially according to Clegg et al. [9]. High-voltage paper electrophoresis on Whatmann 3-MM paper was performed in a "Varso1"-cooled tank (Savant Instruments, Hicksville, U.S.A.) at 30 V/cm for 3 h with a buffer (pH 4.7) of pyridine-acetic acid-water (5:5: 390, by vol.). The solvent for paper chromatography was n-butanol-acetic acid-water-pyridine (15:3: 12: 10, by vol.) (Hills solvent). Peptides were detected by staining the paper with a 0.050/, ninhydrin-acetone solution a t 40 "C for 30 min. The coloured spots were subsequently eluted from the paper with ZOO/, pyridine in water.

A Kinase That Transfers the γ-Phosphoryl Group of GTP to Proteins of Eukaryotic 40S Ribosomal Subunits

“…Although the modified ribosomal proteins cannot be detected on the stained gel, since only 0.4% of the particles are acylated, the shifted proteins have been positively identified by crossreaction with antibodies to the normal proteins (24). A difference of one positive charge (acetylation of the NHrterminal serine) in two otherwise identical E. coli ribosomal proteins (L7 and L12) cause a similar change in position (25). Thus, one can predict with some confidence the behavior of chemically modified proteins.…”

Mechanism and Rates of Exchange of L7/L12 between Ribosomes and the Effects of Binding EF-G

“…coli), L12 is partially N-acetylated to form protein L7 (8). The ratio of L7 to L12 is known to change throughout the growth phase (8)(9)(10) and has been linked to variation in the stability of the stalk complex via hydrogen exchange mass spectrometry (MS) experiments (11).…”

“…The ratio of L7 to L12 is known to change throughout the growth phase (8)(9)(10) and has been linked to variation in the stability of the stalk complex via hydrogen exchange mass spectrometry (MS) experiments (11). L12 CTD and NTD are connected via a flexible hinge region providing mobility to the highly structured CTD (12,13).…”

Isopeptide linkage between N-alpha-monomethylalanine and lysine in ribosomal protein S11 from Escherichia coli.

“…Proteins L16 and L33 are N-a-monomethylated at their methionine and alanine residues, respectively (4)(5)(6)(7), and N-a-acetylation was established for proteins S18 (25) and L7 (26). As yet it is unknown why proteins L16 and L33 are methylated, whereas large variations in the amount of L7 present relative to its nonacetylated form, L12, in the ribosome during the growth cycle suggest some specific control mechanism for the acetylation process (27).…”