An Improved Technique for the Analysis of Ribonucleoprotein Fragments from <i>Escherichia coli</i> 30S Ribosomes

Brimacombe, Richard; Morgan, Joan; Cox, Robert A.

doi:10.1111/j.1432-1033.1971.tb01591.x

Cited by 27 publications

(18 citation statements)

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“…The two 50-S fragments described here fulfil two important criteria for specificity which we have previously discussed [7,15], viz. their proteins are present in equimolar amounts, and the level of contaminating protein (noise) is low.…”

Section: Resultsmentioning

confidence: 99%

“…The onedimensional detergent-gel system has the advantage that many samples can be handled simultaneously, and also that the RNA and protein are dissociated in situ on the gel [15]. On the other hand, the obvious disadvantage of this system is that by no means all of the 50-S proteins can be separated in one dimension.…”

Section: Resultsmentioning

confidence: 99%

“…Fig. 3 also gives the molecular weights of the proteins, estimated from their mobility in the sarkosyl gel, as described previously [15] (and J. Morgan and R. Brimacombe, unpublished results).…”

Section: Resultsmentioning

confidence: 99%

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Two Specific Ribonucleoprotein Fragments from the 50-S Sub-Particle of Escherichia coli Ribosomes

Newton

Brimacombe

1974

Eur J Biochem

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50-S ribosomal particles from Escherichia coli were hydrolysed with ribonuclease TI, and the ribonucleoprotein fragments produced were fractionated on polyacrylamide gel. The proteins contained in these fragments were analysed on gels containing the detergent sarkosyl (N-laurylsarcosine), using the technique already published. Two-dimensional gel electrophoresis was used for the final identification of the proteins.Two very specific fragments were obtained, the smaller containing only proteins L1 and L9, and the larger containing only L5, L18 and L25. The latter proteins have been shown by other authors to be involved in binding to 5-S RNA, but in this case the fragment contained more RNA than can be accounted for by 5-S RNA alone. This offers the possibility of determining the regions of 23-S RNA associated both with these two groups of proteins and with 5-S RNA. Both fragments are discussed in terms of a possible oligonucleotide analysis of their RNA moieties.It has been shown that under suitably controlled conditions 30-S ribosomal sub-particles from Escherichia coli can be digested with nuclease to yield specific fragments of ribonucleoprotein [l, 21. Such fragments have proved useful in several ways. Analysis of their protein content provides information as to the grouping of individual proteins within the ribosome [2], while analysis of the constituent RNA sequences can show which regions of the ribosomal RNA are associated with a particular group of proteins [3]. Further, such fragments have been used in more functionally oriented studies, such as the binding of streptomycin [l].A similar fragmentation approach has been used by other authors on the 50-S sub-particle. Allet and Spahr [4] isolated two large ribonucleoprotein fragments which showed some specificity in their protein content. Kagawa et al. [5] have reported a series of small fragments all from the 50-S sub-particle, but unfortunately were not able to described their proteins in the accepted nomenclature [6]. In this paper we describe the isolation of two small very specific ribo-The preceding paper in this series is by Pongs and Nierhaus (Mol. Gen. Genet., in press).Enzyme. Ribonuclease T, (EC 3.1.4.8).Definition. An A,,, unit is the quantity of material contained in 1 ml of a solution which has an absorbance of 1 at 260 nm, when measured in a l-cm path-length cell.-~ nucleoprotein fragments from the Escherichia coli 50-S ribosome, using the methodology we have already described for the 30-S sub-particle [2,7], extended to include two-dimensional gel electrophoresis [8] for the final identification of the proteins involved. Preliminary experiments have shown that RNA fragments of the expected order of size can be isolated from these RNA . protein complexes, suggesting that both fragments could be usefully subjected to oligonucleotide analysis. MATERIALS AND METHODS Preparation of RibosomesNH,Cl-washed ribosomal sub-particles were prepared as described previously [7], with the exception that E. coli strain A19 was used instead of MRE 600. Rib...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Two Specific Ribonucleoprotein Fragments from the 50-S Sub-Particle of Escherichia coli Ribosomes

Newton

Brimacombe

1974

Eur J Biochem

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“…An unambiguous identification of the labeled proteins was made by polyacrylamide gel electrophoresis run in the presence of the detergent Sarkosyl (18). The results are shown in Fig.…”

Section: Methodsmentioning

confidence: 98%

“…Proteins were isolated as described in Materials and Methods. Sarkosyl-polyacrylamide gel slabs were run essentially by the procedure of Brimacombe et al (18).…”

Section: Discussionmentioning

confidence: 99%

Affinity labeling of the ribosomal decoding site with an AUG-substrate analog.

Pongs¹,

Lanka²

1975

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

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The trinucleotide AUG was condensed at the 5'-end with N-bromoacetyl-p-.aminophenylphosphate.This bromoacetylated AUG analog reacted irreversibly with the mRNA binding site of Escherichia coli 70S ribosomes. After reaction of 70S ribosomes with the AUG analog, labeled 30S subunits could be isolated that were programmed for initiation-factor-dependent binding of fMet-tRNAfMet. This shows that this AUG-affinity label reacted in the decoding site for fMet-tRNAfMet. By combination of sodium dodecyl sulfate-, Sarkosyl-, and ureapolyacrylamide gel electrophoresis the AUG-affinity label was found to be irreversibly bound to ribosomal proteins S4, the ram gene product, and S18.Despite recent advances in our understanding of the function of particular ribosomal proteins in protein synthesis (for a review see ref. 1), several important problems remain. Among these is the role of ribosomal proteins in decoding mRNA. This is placed in the perspective of the number of proteins necessary to initiate, propagate, and terminate translation and their function in moving along the message. One way of studying this essential aspect of the translation process is to identify the ribosomal proteins that are directly involved in this part of protein synthesis. Recently, the technique of affinity labeling was successfully applied in several laboratories in order to elucidate the ribosomal proteins that are implicated in the peptidyltransferase center (2-4). Likewise, affinity labeling should help to answer other questions on the functions of ribosomal proteins. We have synthesized a bromoacetylated AUG analog, which irreversibly reacts at the decoding site of Escherichia coli ribosomes. After chemical linkage of this AUG analog, 30S subunits could be isolated, which were programmed for initiationfactor-dependent binding of fMet-tRNAfMet. This allowed identification of ribosomal proteins associated with decoding of mRNA.MATERIALS AND METHODS Ribosomes of E. coli A19 were isolated and purified as described (5). They were salt-washed once with 1 M NH4Cl.About 60-70% of these ribosomes bound fMet-tRNAfMet in a puromycin-sensitive state and represented tight couples according to the nomenclature of Noll (6). fMet-tRNAfMet was prepared from purified E. coli tRNAfMet (Boehringer, Germany) according to ref. (pH 9.0) buffer. After treatment of the incubation mixture with alkaline phosphatase, nitrophenyl-pA-U was isolated by TEAE-cellulose column chromatography using a 0.01 M-0.2 M NaCl linear gradient. The subsequent enzymatic condensation of nitrophenyl-pA-U with GDP by polynucleotide phosphorylase in the presence of RNase T, followed similar lines. The final nitrophenyl-pA-U-G was catalytically reduced to aminophenyl-pA-U-G and then bromoacetylated as described for the reduction and bromoacetylation of nitrophenyl-dTp (11). A detailed procedure of the synthesis of Nacylated aminophosphorylated oligonucleotides will be published elsewhere.

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Assembly of bacteriophage T4 tail fibers: Identification and characterization of the nonstructural protein gp57

Herrmann

Wood

1981

Molec. Gen. Genet.

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Formation of both the tail fiber and the baseplate of bacteriophage T4 depends on the product of T4 gene 57. A single amber mutation in that gene causes loss of two T4-specific proteins. Their molecular weights are 18,000 and about 6,000, respectively, based on their electrophoretic mobilities in SDS-polyacrylamide gels. E. coli carrying a cloned T4 DNA fragment of about 700 basepairs, which directs the synthesis of the smaller protein only, specifically supports the growth of gene 57 amber mutants. We conclude that the small protein is a functional product of gene 57.

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An Improved Technique for the Analysis of Ribonucleoprotein Fragments from Escherichia coli 30S Ribosomes

Cited by 27 publications

References 14 publications

Two Specific Ribonucleoprotein Fragments from the 50-S Sub-Particle of Escherichia coli Ribosomes

Two Specific Ribonucleoprotein Fragments from the 50-S Sub-Particle of Escherichia coli Ribosomes

Affinity labeling of the ribosomal decoding site with an AUG-substrate analog.

Assembly of bacteriophage T4 tail fibers: Identification and characterization of the nonstructural protein gp57

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