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Two isogenic derivatives of Escherichia coli K12: strains EA 1 (Met-, Biot-, RCstr and EA 2 (Mei-, Biot-, RCrel) when grown in the presence of ethionine in place of methionine, synthesize ribosomes whose RNA is submethylated and whose proteins contain ethionine. Net ribosome synthesis in the presence of ethionine is greater in the RCrel strain than in the RCstr strain. The precedinig paper describes results obtained with the latter strain. Here we present further studies carried out with the isogenic RCrel strain EA 2 As in the case of the RCstr strain (previous paper) ribosomes synthesized by the RCrel strain grown in the presence of ethionine display defective association properties. A large fraction of these "ethionine" ribosomes is found as free subunits in crude extracts containing 10 mM Mg+ prepared from strain EA 2 grown in an ethionine-containing medium. These free "ethionine" subunits have sedimentation coefficients of 28 S and 45 S approximately while the "ethionine" subunits found associated as 70 S particles in crude bacterial extracts sediment at 30 S and 50 S respectively. Both classes of "ethionine" subunits (free, and associated) can be methylated in vitro in the presence of S-adenosyl methionine with or without the addition of an external source of methylases (100000 x g supernatant of a homologous crude bacterial extract) The latter observation shows that the "ethionine" subunits contain bound methylases. These enzymes do not seem to be normal ribosomal proteins nor to be bound specifically to "ethionine" ribosomes since (a) they are removed by washing the "ethionine" ribosomes with 1 M NH4Cl, and (b) they are found bound to normal methionine ribosomes from which they are also removed by washing with 1 M NH4Cl. [ABSTRACT FROM AUTHOR]

This paper describes three types of experiments which indicate that the binding sites for elongation factor 1 (EF-1) and elongation factor 2 (EF-2) on ascites cell ribosomes are not identical and perhaps not even overlapping. The experimental evidence presented includes direct competitive binding of labeled elongation factors to ribosomes as well as the influence of pokeweed antiviral protein and Escherichia coli anti-L7/L12 proteins on the binding and function of the two factors. It is further shown that EF-1β from Artemia salina does not function in displacing EF-1 from mouse ascites tumor cell ribosomes. These results also support our recently proposed model that EF-1 remains bound to the ribosome during the peptide chain elongation cycle. [ABSTRACT FROM AUTHOR]

Proteins L6 and L29 in the 60-S subparticle of mammalian ribosomes interact in situ under gentle conditions by intermolecular disulfide bond formation. For identifying the contact regions of the two proteins the disulfide complex was isolated from whole ribosomes by preparative polyacrylamide gel electrophoresis and subjected to cyanogen bromide cleavage. For effective cleavage non-oxidizing conditions had to be maintained throughout the preparation. A simple and generally applicable method of high-efficiency gel pre-electrophoresis with anionic and cationic thiols was developed. Under these conditions reversibly cross-linked CNBr fragments of L6 and L29 could be isolated in high yield (Mr approx. 13000 and 7000, respectively). After [14C]carboxymethylation of the reduced disulfide links smaller contact sequences were obtained by pepsin digestion and characterized by two-dimensional peptide mapping. These smaller contact peptides were contained within the corresponding CNBr fragments. Both contact peptides were hydrophilic and relatively basic. [ABSTRACT FROM AUTHOR]

The bifunctional reagent ethyl 4-azidobenzoylaminoacetimidate was used to crosslink specifically ribosomal protein S1 to 16-S RNA within 30-S subunits. The reagent was attached to isolated protein S1. The modified protein was reassociated with protein-S1-depleted 30-S subunits and then crosslinked to the RNA molecule. The covalently bound 16-S RNA protein S1 complex was isolated and the RNA fragment C-U-A-A-G-G-G-G-U-U-A-A-G-U-C-G-A-C-C-G-C-C-U-G-G-G-G-A-G (positions 861-889) was characterized to be crosslinked to protein S1. [ABSTRACT FROM AUTHOR]

Protein L11 was isolated from the 50-S subunit of Escherichia coli ribosomes, using two salt extractions and two chromatographic separations on CM-cellulose. The unusual behavior of the protein when run on sodium dodecyl sulfate electrophoresis showed multiple bands. The complete primary structure of protein L11 is presented in detail. Its sequence was derived from peptides obtained by digesting the protein with trypsin, chymotrypsin, thermolysin, Staphylococcus aureus protease and, after modification, with trypsin. Chemical cleavage was performed with cyanogen bromide. Sequencing of the various peptides was achieved by manual micro-dansyl-Edman degradations and automatic methods. The N-terminal residue of the protein is blocked and was not degradable in the liquid-phase sequenator by the Edman method. It was identified by a combination of enzymatic cleavage and mass spectrometry. Protein L11 contains three methylated amino acid residues, a Nα-trimethylalanine, and two residues of Nϵ-trimethyllysine. Their behaviour and influence in the sequence elucidation are described. The protein contains 141 amino acid residues and has a molecular weight of 14874. Secondary structure predictions of the protein are given, and its sequence is compared with those of other E. coli ribosomal proteins. [ABSTRACT FROM AUTHOR]

The effects of ultraviolet irradiation on Escherichia coli 30-S ribosomal subunits were studied. At the doses of radiation used in this work (0–4.5 ⊗ 105 quanta/30-S subunit), only protein S7 was found to be significantly crosslinked to the 16-S RNA. In conditions where 25% of the protein was covalently crosslinked, the ability of the irradiated 30-S subunits to reassociate with 50-S subunits and their activity in polyphenylalanine synthesis decreased strongly. Similar results were obtained by irrradiation with a germicide lamp (254 nm) or with a monochromatic ultraviolet light at 248 nm. No additional proteins were crosslinked to the 16-S RNA by irradiating 30-S subunits depleted in protein S1 or 70-S ribosomes. The covalent complex of 16-S RNA and protein S7 was isolated and digested by T1 ribonuclease. The oligonucleotide remaining attached to the crosslinked protein was characterised as A-C-C-U-C-G [position 1261–1266, see the sequence published by Carbon et al. (1979) Eur. J. Biochem. 160, 399–410]. Analysis of this fragment suggests that protein S7 was linked to the cytosine at position 1265 in the RNA sequence. [ABSTRACT FROM AUTHOR]

Analyses are described of three types of ribosomal fragment, all derived from the well-characterized ribonucleoprotein species consisting of proteins S7, S9, S10, S14 and S19, together with RNA from sections O′-D-E′-K-P-P′-E-A of the 16-S sequence. I. When 30-S subunits were hydrolysed with ribonuclease T1 in the presence of deoxycholate in addition to the components previously described, a fragment could be isolated which contained only proteins S7 and S 19~ together with minor amounts of S 13 or S 14. Oligonucleotide analysis of this fragment showed that it contained RNA from sections O′-E′ and P-A of the 16-S RNA~ but that section K (from the middle of this area) was missing. 2. It has previously been shown that when 30-S subunits are irradiated with ultraviolet light, protein S 7 is the primary target of cross-linking of protein to RNA. By making use of this reaction, ribonucleoprotein fragments were isolated from irradiated 30-S subunits, the unbound proteins were removed, and RNA fragments containing covalently linked protein S 7 were identified. It was possible to demonstrate that the site of cross-linking lies within the O′-A region of the 16-S RNA, and more precise experiments showed that this site almost certainly is in the P-A region. 3, When the parent five-protein ribonucleoprotein fragment (above) was deproteinized under very mild conditions, RNA complexes could be isolated which consisted of non-contiguous sequences~ but which migrated as a single species into a polyacrylamide gel. Analysis of one of these complexes showed that it contained sequences from sections O′-E′ and P-A, but that section K was missing (cf. first paragraph above). This demonstrated that these two separate regions of RNA interact within the 30-S subunit, independently of the presence of protein. [ABSTRACT FROM AUTHOR]

The activity of a 30-S CsCl core lacking proteins S1, S2, S3, S5, S9, S10, S14, S20 and S21 has been studied in the ribosome-dependent GTPase reactions in the presence of the 50-S subunit with and without methanol. Without methanol, the 30-S CsCl core was unable to sustain GTPase activity dependent on elongation factor G (EF-G), while it was only slightly active in the presence of elongation factor T (EF-T). With EF-T, addition of methanol induced in the presence of either 30-S subunits or 30-S CsCl cores an activity which was more than 10-fold higher than that observed with the 30-S subunit in the absence of methanol. Methanol lowered the Mg2+ optimum of the EF-T- dependent GTPase reaction from approximately 20 mM to approximately 10 mM. In the absence of methanol the EF-G-dependent GTPase reaction at low concentration of monovalent cations depends on the 50-S subunit alone (30-S-uncoupled EF-G GTPase). Addition of the intact 30-S subunit but not of its CsCl core abolished inhibition of the 30-S-uncoupled EF-G GTPase by NH4+. The 30-S CsCl core caused the same effect as the 30-S subunit when methanol was present. 30-S-uncoupled EF-G GTPase activity was lower than the GTPase activity dependent on 30-S plus 50-S subunits at [EF-G]/[50-S] below 5 but was considerably higher in the presence of a large excess of EF-G. In the presence of methanol the 30-S CsCl core behaved similarly to the 30-S subunit. Our results indicate that the action of the 30-S subunit in elongation-factor-dependent GTPases is supported by structural features that are preserved in the 30-S CsCl core. The 30-S split proteins are therefore not essential for EF-G and EF-T activities in the hydrolysis of GTP. With EF-T, in all conditions tested association of the ribosomal subunits appeared to accompany GTPase activity. Association seems also to be a prerequisite of the EF-G GTPase activity that depends on both ribosomal subunits. [ABSTRACT FROM AUTHOR]

A method is presented to isolate in pure form two acidic proteins, indicated as A1- and A2-protein from 50-S ribosomes of Escherichia coli. The advantage of this method is that it does not require prior isolation of subunits. The molecular weight of A1- and A2-protein was found by dodecylsulfate gel electrophoresis to be 11 500 with a maxum uncertainty of 1500. Sedimentation equilibrium centrifugation in 5 M guanidine hydrochloride indicates a molecular weight in the range of 12 000-13 000. The amino acid composition of the two proteins is remarkable and indistinguishable. A-protein has a high content of alanine and does not contain tyrosine, tryptophan, histidine or cysteine. On gel-electrofocusing the isoelectric points of A1- and A2-protein are slightly different, namely pH 4.7 and pH 4.85. The amount of A-protein in actively diving cells indicates that contrary to 30-S ribosomes, 50-S ribosomes have a restricted element of repeat. The number of copies of A-protein is found to be somewhat larger than two. [ABSTRACT FROM AUTHOR]

1. The 43 S precursor of the 50 S ribosomal subunit shows a protein pattern very similar to that of a “core” particle derived from 50 S subunits on CsCl gradients. 2. A comparison of the protein patterns of “core” particles from 50 S subunits after CsCl equilibrium runs or incubation with increasing LiCl concentrations over the molarity range 0.4-6 M revealed that the proteins are successively released from the “core” in five distinct groups. 3. The protein pattern of the 21 S precursor of 30 S ribosomal subunits was compared with that of the reconstituted intermediate particle and LiCl-treated “core” particles of 30 S subunits. The 21 S is similar to both the reconstituted intermediate and the “core” particles derived by a 1 M LiCl treatment, whereas the reconstituted intermediate and this core particle differ rather more. [ABSTRACT FROM AUTHOR]