The Ribosome of Escherichia coli

Brimacombe, Richard; Nierhaus, K H; Ra, Garrett; Hg, Wittmann

doi:10.1016/s0079-6603(08)60585-1

Cited by 60 publications

(20 citation statements)

References 219 publications

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“…Additional information can be obtained from positions of binding sites of ribosomal proteins on the primary structure of 16S RNA (reviewed in ref. 28) and from the results of RNase fragmentation of the 30S particles (1,28). These results permit grouping of the ribosomal proteins as shown in Fig.…”

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confidence: 59%

“…First, it is likely that protein S4 occupies some central position in the 30S particle: (i) it is distinguished by the most reported crosslinks and interactions with other proteins (4, 6, 10, 28); (ii) it has no antigenic determinants on the surface of the particle and thus is buried inside (30); (iii) it is bound simultaneously with several extended but noncontiguous regions of the 16S RNA chain (28,31,32). The central position and the unique binding of globular protein S4 to remote long sections of the RNA chain could be realized in the most natural way if it was positioned between the two branches of the Y-shaped RNA molecule, interacting with both the branches.…”

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confidence: 99%

See 1 more Smart Citation

Quaternary structure of the ribosomal 30S subunit: model and its experimental testing.

Spirin¹,

Serdyuk²,

Shpungin³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

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In considering the structure of the 30S subunit of the Escherichia coli ribosome, we have assumed that: (l) all or almost all the proteins within the 30S particle are compact and globular, as recently shown for the isolated proteins S4, S7, S8, S15, and S16 in solution [Serdyuk, I. N., Zaccai, G. & Spirin, A. S. (1978) In recent years, the technique of chemical crosslinking of neighboring proteins by bifunctional reagents and measurements of the distances between deuterated or fluorescent-labeled proteins by using neutron scattering or energy transfer techniques, respectively, have contributed much to the knowledge of the protein topography in ribosomal particles and especially in the ribosomal 30S subunit of Escherichia coli. From these and a number of less direct data, several models of the topography of ribosomal proteins in the 30S particle have been proposed (1)(2)(3)(4)(5)(6)(7)(8). Independently, the use of the immunoelectron microscopy technique has also led to two different models of the topography of ribosomal proteins on the surface of the 30S particle (9-12; see also ref. 13).However, two complications have hampered further progress. First, no information was available as to three-dimensional structure and morphology of the 16S RNA; this RNA is known to be a structural backbone for arrangement of ribosomal proteins. Second, immunoelectron microscopy (9, 10) and neutron scattering (14, 15) data on proteins within the 30S subunit, as well as some physical measurements of isolated ribosomal proteins (16-21), have suggested strongly elongated or very expanded shapes of many ribosomal proteins; this led to ambiguity in interpretation of the results on chemical crosslinking, energy transfer, etc.Most recently, a specific compact conformation of the 16S RNA has been visualized by electron microscopy (22). On the other hand, a number of ribosomal proteins, such as S4, S7, S8, S15, and S16, have been reinvestigated and found to be compact globular proteins with cooperative tertiary structures (23)(24)(25). Based on these new results and using the numerous data reported on mutual arrangement (topography) of ribosomal proteins, we have constructed a model of the quaternary structure of the ribosomal 30S particle and then checked it by neutron and x-ray scattering experiments.MODEL CONSTRUCTION Initial Assumptions. In constructing the model, we have adhered to the size and the asymmetric three-dimensional structure of the ribosomal 30S subunit first deduced by one of us from electron microscopy of freeze-dried and shadow-cast samples of the particles (26). Morphologically, the 30S particle was found to be subdivided into a "head," a "body," and a side "bulge" (Fig. 1). A similar subdivision of the 30S particles, with a side "platform," was reported also by Lake and Kahan (11) and Lake (27) on the basis of electron microscopy of negatively stained samples.We have proceeded from two principal assumptions: (i) RNA within the 30S particle has a specific V-like or Y-like conformation (Fig. 1), as it is visu...

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confidence: 59%

mentioning

confidence: 99%

Quaternary structure of the ribosomal 30S subunit: model and its experimental testing.

Spirin¹,

Serdyuk²,

Shpungin³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

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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%

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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“…The earlier studies (reviewed in refs. 2 and 3; see also refs. 4-7) used various procedures in the presence or absence of tRNA to modify the ribosome, but, in general, these methods were not able to distinguish between a direct action of the reagent on the binding site and an indirect effect due to a modification elsewhere.…”

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confidence: 93%

Localization of the decoding region on the 30S Escherichia coli ribosomal subunit by affinity immunoelectron microscopy.

Keren-Zur¹,

Boublík²,

Ofengand³

1979

Proc. Natl. Acad. Sci. U.S.A.

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The decoding region of the Escherichia coli ribosome has been localized by affinity immunoelectron microscopy. Valyl-tRNA al, derivatized at the a-amino end with the dinitrophenyl group, was bound to the ribosomal P site of 70S ribosomes and crosslinked specifically to 16S RNA by 310-to 325-nm irradiation. Previous work has shown that the crosslink occurs between the 5' anticodon base of the tRNA and a pyrimidine in the 3' one-third of the 16S RNA. We recently observed that Eschertchia coli AcVal-tRNAlal (and certain other tRNAs) bound at the P site of 70S ribosomes could be covalently crosslinked with high efficiency to 16S ribosomal RNA by irradiation at 310-325 nm (13). This reaction linked the anticodon loop of the tRNA to the 3' one-third of the 16S ribosomal RNA (15,16). Since the tRNA was covalently attached via its anticodon to the presumptive decoding region of the ribosome, the possibility of visualization of this site by EM was considered. Preliminary experiments showed that it was not possible to directly visualize tRNA on the ribosome with the present EM methods. We decided, therefore, to attach a suitable antigenic determinant to the tRNA in order to use antibody (Ab) against it as a marker for the location of the tRNA molecule. This technique, which we call affinity immuno-EM, has provided a direct approach to localization of the decoding region on the 30S particle. MATERIALS AND METHODSN-(y-Dinitrophenyl)aminobutyryl (DNP-AbuX3HJVal-tRNA. DNP-Abu (Sigma) was coupled to N-hydroxysuccinimide (HONSu) with N,N'-dicyclohexylcarbodiimide in dimethylformamide. Esterification was followed by silica gel thin-layer chromatography (Eastman) developed with ethyl acetate/methanol (10:1). The RF of DNP-Abu = 0.1; RF Of DNP-Abu-ONSu = 0.9. After isolation, the product was tested by reaction with glucosamine. There was complete conversion to a new product, possessing an RF of 0.48 (the RF of DNP-Abu was 0.86 and the RF of DNP-Abu-ONSu was 0.9) when analyzed by silica gel thin-layer chromatography developed with chloroform/methanol/HOAc (65:30:5 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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The Ribosome of Escherichia coli

Cited by 60 publications

References 219 publications

Quaternary structure of the ribosomal 30S subunit: model and its experimental testing.

Quaternary structure of the ribosomal 30S subunit: model and its experimental testing.

Binding of Ribosomal Proteins to RNA Covalently Coupled to Agarose

Localization of the decoding region on the 30S Escherichia coli ribosomal subunit by affinity immunoelectron microscopy.

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