Search PubMed⌕ Search

Biomedical subjects

R Brimacombe

Publications and source records attributed to R Brimacombe.

At least 127 records · Page 7Linked to original sources
PubMed1977

Characterisation of RNA fragments obtained by mild nuclease digestion of 30-S ribosomal subunits from Escherichia coli.

When Escherichia coli 30-S ribosomal subunits are hydrolysed under mild conditions, two ribonucleoprotein fragments of unequal size are produced. Knowledge of the RNA sequences contained in these hydrolysis products was required for the experiments described in the preceding paper, and the RNA sub-fragments have therefore been examined by oligonucleotide analysis. Two well-defined small fragments of free RNA, produced concomitantly with the ribonucleoprotein fragments, were also analysed. The larger ribonucleoprotein fragment, containing predominantly proteins S4, S5, S8, S15, S16 (17) and S20, contains a complex mixture of RNA sub-fragments varying from about 100 to 800 nucleotides in length. All these fragments arose from the 5'-terminal 900 nucleotides of 16-S RNA, corresponding to the well-known 12-S fragment. No long-range interactions could be detected within this RNA region in these experiments. The RNA from the smaller ribonucleoprotein fragment (containing proteins S7, S9 S10, S14 and S19) has been described in detail previously, and consists of about 450 nucleotides near the 3' end of the 16-S RNA, but lacking the 3'-terminal 150 nucleotides. The two small free RNA fragments (above) partly account for these missing 150 nucleotides; both fragments arose from section A of the 16-S RNA, but section J (the 3'-terminal 50 nucleotides) was not found. This result suggests that the 3' region of 16-S RNA is not involved in stable interactions with protein.

Base Sequence↗
PubMed1976

RNA sequences associated with proteins L1, L9, and L5, L18, L25, in ribonucleoprotein fragments isolated from the 50-S subunit of Escherichia coli ribosomes.

32P-labelled 50-S subunits from Escherichia coli ribosomes were hydrolysed under conditions known to give rise to two specific ribonucleoprotein fragments, containing proteins L1, L9, and L5, L18 and L25 respectively. RNA corresponding to these ribonucleoproteins was isolated and purified, and the various RNA fragments obtained were subjected to oligonucleotide analysis. The results showed that the RNA associated with proteins L1 and L9 was very similar to the RNA found with protein L1 after controlled digestion of 23-S-RNA - L1 complexes (described elsewhere); this RNA lies within a region 550-1000 nucleotides from the 3' terminus of 23-S RNA. The RNA associated with proteins L5, L18, and L25 consisted predominantly of two species of similar size. One was 5-S RNA, and the other a fragment of 23S RNA, lying within the region 450-1000 nucleotides from the 3' terminus.

Alkaline Phosphatase↗
PubMed1976

Studies on the environment of protein S7 within the 30-S subunit Escherichia coli ribosomes.

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. 1. When 30-S subunits were hydrolysed with ribonuclease T1 in presence of deoxycholate in addition to the components previously described, a fragment could be isolated which contained only proteins S7 and S19, together with minor amounts of S13 or S14. 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 S7 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 S7 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.

Bacterial Proteins↗
PubMed1975

Specific cross-linking of proteins S7 and L4 to ribosomal RNA, by UV irradiation of Escherichia coli ribosomal subunits.

Radioactive 30S and 50S subunits from E. coli ribosomes were irradiated with UV light, under conditions giving rise to approximately 10% cross-linking of protein to ribosomal RNA. Irradiation to levels of cross-linking higher than 10% caused unfolding of the ribosomal subunits, even in the presence of 5 mM magnesium. The specificity of the cross-linking reaction at this low level was found to be extremely high. Cross-linked RNA-protein complexes, freed from unbound protein, were treated with nuclease and then analysed on Sarkosyl gels. S7 was found to be the primary target of the cross-linking reaction in the 30S particle. This was proven by using subunits from both E. coli MRE 600 and A19, whose respective S7 species differ markedly. In the 50S particle, L4 was the primary target, although L2 was also cross-linked to a small extent. Ambiguity in the identification of L4 in the Sarkosyl system was resolved by two-dimensional electrophoresis, which was also used to demonstrate a genuine linkage to RNA in the case of both S7 and L4; proteins spots containing 32P were observed, derived from these two proteins, when subunits containing 32P-RNA were irradiated, treated with nuclease, and applied to the electrophoresis. The identities of S7, L4 and L2 were finally confirmed by Ouchterlony tests with protein-specific anti-sera.

Electrophoresis, Polyacrylamide Gel↗
PubMed1975

Nucleotide sequences of Escherichia coli 16-S RNA associated with ribosomal proteins S7, S9, S10, S14 and S19.

1. A ribonucleoprotein fragment containing proteins S7, S9, S10, S14, and S19 was isolated in high yield from Escherichia coli 30-S ribosomal sub-particles. The same fragment was obtained whether ribosomes from E. coli strain A19 or MRE 600 were used, despite the fact that protein S7 differs widely between the two strains. RNA was extracted from this fragment and fractionated on gels containing 7 M urea, to reveal "hidden breaks". A well-defined and reporducible pattern of RNA fragments was obtained, with the main components being approximately 300, 240, 130, 115 and 75 nucleotides in length, respectively. The pattern of RNA fragments obtained was also independent of the strain of E. coli used. 2. Two-dimensional fingerprints were made from ribonuclease T1 hydrolysates of these RNA fragments, labelled with 32P, and the oligonucleotides were further analysed by digestion with either ribonuclease A or U2. The data obtained were fitted in detail to the new 16-S RNA sequence map of Ehresmann et al. (1975). Again no significant differences were observed between the RNA from E. coli A19 or MRE 600. The RNA sequences found lay in the region O'-D-E'-K-P-P'-E-A of the 16-S RNA, with a clear excision of several nucleotides in section E'-K. The total sequence length covered was approximately 430 nucleotides.

Base Sequence↗