Active sites in <i>Escherichia coli</i> ribosomes

Pongs, Olaf; Nierhaus, Knud H.; Erdmann, Volker A.; Wittmann, H. G.

doi:10.1016/0014-5793(74)80686-1

Cited by 79 publications

(29 citation statements)

References 117 publications

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“…We interprete this result that pyrophosphoryl transferase and elongation factor Tu can react simultanoulsy on the ribosomes, Our data indicate that the E. coli pyrophosphoryl transferase binds to a site different from that of the elongation factors. These findings are in good agreement with previous experiments in which it was shown that the two acidic proteins L7/L12 from the large ribosomal subunit are not required for the pyrophosphoryl transfer reaction [ 17,18], whereas they are for EF-Tu and EF-G functions [1,2]. The possibility that transferase and elongation factors do not bind to the same but rather to different ribosomes is unlikely, because ribosomes precharged with EF-Tu or EF-G were inactive in the pyrophosphoryl transferase-dependent formation of pppGpp and ppGpp but active in binding the transferase [16].…”

Section: Resultssupporting

confidence: 93%

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The binding of the pyrophosphoryl transferase and the elongation factor Tu and G to ribosomes from Escherichia coli

Kleinert

Richter²

1975

FEBS Letters

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Section: Resultssupporting

confidence: 93%

“…It has been shown that the peptide chain elongation factor Tu (EF-Tu) and G (EF-G) from bacteria interact on ribosomes at a common region (for reviews see [1,2]). The existence of such a region has been demonstrated by competition experiments where ribosomes carrying EF-G are inactive in the EF-Tudependent Phe-tRNA binding reaction.…”

Section: Introductionmentioning

confidence: 99%

The binding of the pyrophosphoryl transferase and the elongation factor Tu and G to ribosomes from Escherichia coli

Kleinert

Richter²

1975

FEBS Letters

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“…Recent evidence indicates that all but S6 also bind directly to 16-S RNA when the latter is prepared by an acetic acid/urea method [lo]. Numerous investigations have demonstrated that the above proteins and others are important for initiationfactor-dependent binding of met-tRNA or EF-Tudependent binding of aminoacyl tRNA to the ribosome (see [27] and [50] for reviews). However, it should be borne in mind that the binding of tRNA in these experiments may be affected indirectly by such factors as the inhibition of mRNA or EF-Tu binding or subunit association.…”

Section: Ajjin Ity Chroma Togruphy ~F E Coli 30-s Ribosomal Proteinsmentioning

confidence: 99%

“…1970. as inactivation of ribosomal proteins or RNA by chemical modification [15-171, inhibition of a specific functional activity by reaction with antibodies directed against selected ribosomal proteins [ 18,191 and affinity labeling of ribosomal components physically situated at a specific binding site . A summary of some of this work is available [27]. From a correlation of these data one can infer that a surprisingly large number of ribosomal proteins from both subunits may be involved in interactions with extraribosomal RNA and protein factors during translation.…”

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confidence: 99%

Binding of Ribosomal Proteins to RNA Covalently Coupled to Agarose

Burrell

Horowitz

1977

Eur J Biochem

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An affinity chromatographic procedure, using ribosomal or tRNA covalently coupled to agarose through an adipic acid dihydrazide spacer, has been developed to isolate and identify the Escherichia coli ribosomal proteins which specifically bind to these RNA molecules. Transfer RNA, 5-S, 16-S and 23-S ribosomal RNA were readily immobilized on agarose-dihydrazide: the maximum amount coupled decreasing with increasing molecular size. Specific binding of ribosomal proteins to RNA was observed in buffer containing 0.3 M KCI and 0.02 M MgC12. Bound proteins were eluted with a high-salt, 2 M KCI, buffer containing 0.005 M EDTA. Two ribosomal proteins, identified as L18 and L25 by two-dimensional gel electrophoresis, bound tightly to E. cob 5-S RNA;' small amounts of a third protein, L5, also were bound. These proteins did not bind to immobilized tRNA nor did 30-S ribosomal proteins bind to 5-S RNA. Denatured 5-S RNA bound less L l 8 and L25 than the native species. Several 30-S and 50-S ribosomal proteins bind to immobilized tRNA. The major 5 0 3 subunit proteins tightly bound to tRNA were identified as L3, L4, L5, L7 and L8/L9. Smaller amounts of proteins L1, L2, L11, L16 and L21 were also observed. Proteins L2 and L16 were retarded by agarose-bound tRNA. Protein S3 was the major 30-S subunit protein bound to tRNA. Lesser quantities of proteins S6, S9, S13 and S18 also interacted with tRNA.Studies of ribosomal protein -RNA interactions can give important information on the role of individual components in the structure and function of the ribosome. The discovery that the procaryotic ribosome can be self-assembled in vitvo [1,2] and the continuing elucidation of the highly intricate reaction sequence of protein biosynthesis, involving reversible associations of the ribosome with various exogenous molecules such as messenger RNA, transfer RNA, and nucleotides, suggest u pviovi that protein -protein and protein -nucleic acid interactions are essential in these complex macromolecular processes. Support for this supposition has come from several experimental approaches. Reconstitution experiments using purified ribosomal constituents have permitted a tentative identification of the ribosomal proteins that selectively bind to 5-S RNA [3-51, 16-S RNA [6-lo], and 2 3 4 RNA [ l l -141 and thus presumably are those vital to the structural integrity of the ribosome. Moreover, an impressive amount of data delineating functional sites on the ribosome has accumulated from recent investigations. utilizing such approaches From a correlation of these data one can infer that a surprisingly large number of ribosomal proteins from both subunits may be involved in interactions with extraribosomal RNA and protein factors during translation. The results, however, have not allowed an unambiguous identification of the elements comprising ribosomal active sites, perhaps owing to the indirect nature of these probes and to difficulties in interpretation caused by the high degree of cooperativity inherent in situ.In an attempt to develop an alterna...

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“…In Escherichia coli, a protein in the large subunit of ribosome exists in both a-N-acetylated (L7) and unmodified (L12) forms [4,5]. This protein is involved in all the factor-dependent GTP hydrolysis steps carried out by the ribosome during protein biosynthesis (reviewed in [6]). …”

Section: Introductionmentioning

confidence: 99%