A protein-nucleic acid crosslink in 30s ribosomes

Kenner, R. A.

doi:10.1016/0006-291x(73)90016-8

Cited by 91 publications

(22 citation statements)

References 10 publications

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“…f) The fact that S1 and $21 are close neighbors on our model correlates well with the fact that S1 can be crosslinked to the 3' end of the 16S RNA (13) and that $21 is missing in Colicin-treated 30S particles where 16S RNA lacks the 3'…”

supporting

confidence: 75%

Spatial configuration of ribosomal proteins: A computer-generated model of the 30S subunit

Bollen

Cedergren

Sankoff

et al. 1974

Biochemical and Biophysical Research Communications

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supporting

confidence: 75%

Spatial configuration of ribosomal proteins: A computer-generated model of the 30S subunit

Bollen

Cedergren

Sankoff

et al. 1974

Biochemical and Biophysical Research Communications

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“…The technique of reductive crosslinking has been similarly used previously to identify proteins located near the 3' terminus of oxidized 16S RNA in 30S ribosomes of E. coli (27)(28)(29). However, this relatively simple procedure has not found general acceptance for analyzing nucleic acid-protein interactions, possibly because, under some conditions, yields of stable complexes are low.…”

Section: Discussionmentioning

confidence: 99%

Reovirus mRNA can be covalently crosslinked via the 5' cap to proteins in initiation complexes.

Sonenberg¹,

Shatkin²

1977

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

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Proteins that are located adjacent to the 5' end of mRNA in initiation complexes have been detected by chemical crosslinking. Reovirus mRNA containing radioactivity exclusively in the [3H]methyl-labeled "cap," m7G(5')ppp(5') GM, was oxidized with sodium periodate to conveit the 2',3'-cis-diol of the 5'-terminal m7G to a reactive dialdehyde. Oxidized mRNA was incubated in cell-free protein-synthesizing systems derived from wheat germ or mammalian cells, and the resulting mRNA-ribosome initiation complexes were reduced with NaBH3CN. By this chemical procedure, putative Schiff bases between mRNA 5' termini and amino groups of neighboring proteins were stabilized by reduction, yielding covalently linked protein-RNA conjugates. Under conditions of ribosome binding, a limited number of polypeptides associated with the mRNA-ribosome complexes were crosslinked, suggesting that these proteins are positioned near and may interact with the 5' end mRNA during initiation. This method should also be useful for studying the spatial relationships between molecules in other similar nucleoprotein complexes. A "cap" structure, m7GpppX, is present at the 5' end of most eukaryotic mRNAs (1). Several lines of evidence indicate that the cap in reovirus mRNA and other viral and cellular mRNAs has an important functional role. Reovirus capped and methylated mRNA (5'-m7GpppGm) is translated more efficiently than unmethylated (5'-GpppG) or unblocked (5'-ppG) viral mRNAs (2). Removal of the m7G from methylated mRNA by chemical (3) or enzymatic treatment (4) lowers its template activity in cell-free protein-synthesizing systems, and the absence of a 5'-blocked structure results in a decrease in the stability of reovirus mRNA (5). In addition, compounds structurally related to the cap-e.g., m7GMP and m7GDP-inhibit translation in vitro (6-9). These "cap analogs" interfere with the binding of mRNA to 40S ribsomal subunits in initiation compexes, presumably by competing with the 5' end of mRNA for some essential site or component(s).Previously it was reported that wheat germ 40S ribosomal subunits bind to the 5'-terminal region of reovirus mRNAs and protect cap-containing sequences against RNase digestion (10). Because initiation complexes include protein factors capable of binding mRNA, protection of 5' sequences could be due to an interaction of one or more of these proteins with mRNA at or near the cap. Proteins that bind to m7GpppGm-C, the 5'-terminal oligonucleotide derived from reovirus mRNA by T2 RNase digestion, have been detected in high-salt washes of Artemia salina ribosomes (11). Furthermore, the binding of rabbit reticulocyte initiation factor M3 (eIF-4B) to histone mRNA is inhibited by m7GMP, suggesting that binding involves the cap (12). As part of an effort to understand how the cap influences eukaryotic mRNA function, we have assayed initiation complexes for the presence of proteins that are locatedThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore...

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“…This mechanism has been suggested for several DNA denaturing proteins (38). However (14,15). It is thought that this end, unusually rich in pyrimidine residues (39)(40)(41)(42), forms a basepaired complex with mRNA during initiation of protein synthesis; such a complex has been shown to exist with R17 RNA (43).…”

Section: Methodsmentioning

confidence: 99%

“…Detailed structure-function studies have suggested the involvement of S1 in the binding of messenger RNA (9,10), and there are indications that it is important in a ribosomal function involving 3OS-50S subunit interaction (11). Previous studies have implied that S1 binds to RNA molecules containing polypyrimidine sequences (12,13), and this property may be related to its ability to bind to the 3' terminus of 16S RNA (14,15). The choice of model systems used in these studies reflects the observed interactions between S1 and pyrimidine-containing polynucleotides.…”

mentioning

confidence: 99%

Alteration of polynucleotide secondary structure by ribosomal protein S1.

Bear¹,

Ng²,

Derveer³

et al. 1976

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

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Ribosomal 30S protein S1 causes disruption of the secondary structure of certain pyrimidine-containing polynucleotides. Helical poly(U), poly(C,U), and neutral and acidic poly(C) are stoichiometrically converted by SI to structures indistinguishable from their partially or completely thermally denatured forms, as revealed by circular dichroism. Of the several double-and triple-stranded helical polynucleotides tested that contain one polypurine strand and at least one polypyrimidine strand, only the conformation of the DNA-RNA hybrid, poly(A~poly(dT), is perturbed. In the presence of SI, this hybrid undergoes a transition to a new structure that has a circular dichroism spectrum unlike either the native or thermally denatured forms. Intercalated ethidium bromide is released from poly(A)poly(dT) by SI, confirming the occurrence of a conformational rearrangemenit. The translation inhibitor, aurintricarboxylic acid, completely inhibits the action of SI on polypyrimidines, but has no effect on the conformational perturbation of poly(A)'poly(dT). The possible relation between these observations and the biological function of protein SI is discussed.Assignment of specific functional roles to the macromolecular components of the ribosome remains one of the most challenging problems of molecular biology. It can be said that none of the 57 ribosomal components may unambiguously be assigned a particular function in protein synthesis. The reason for this uncertainty is that the structural and functional properties of the ribosome depend to a high degree on cooperative interactions between the various constituent molecules. It would be of great utility in studies of this kind to be able to observe the functional properties of a single ribosomal macromolecule in the presence of all the others. Although such an experiment is impossible in principle, it might be reasonable to approach the situation with model systems containing, for example, a single ribosomal component and one other molecular species of defined structure and properties. Such a model system would be analogous to enzyme-substrate systems in which complex macromolecular substrates are replaced by simple molecules with predictable properties. Any model system suffers from the limitation of having a significantly different structure from the authentic biological system, but nevertheless may yield important clues about the function of a molecular structure that is otherwise too complex to study.We have chosen to study as a model system the interaction of ribosomal protein S1 with polynucleotides. Protein S1 is of considerable interest since it has been found to be part of the replicase enzyme of QB bacteriophage (1), and has been shown to be identical to a protein that selectively inhibits translation in vitro (2-4), in addition to its presence in 30S subunits of ribosomes. Although S1 has been reported to reside in only a small fraction of 30S ribosomes (5, 6), recent studies have shown that it is present in all 70S ribosomes actively engaged in the polymer...

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A protein-nucleic acid crosslink in 30s ribosomes

Cited by 91 publications

References 10 publications

Spatial configuration of ribosomal proteins: A computer-generated model of the 30S subunit

Spatial configuration of ribosomal proteins: A computer-generated model of the 30S subunit

Reovirus mRNA can be covalently crosslinked via the 5' cap to proteins in initiation complexes.

Alteration of polynucleotide secondary structure by ribosomal protein S1.

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