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Titolo:
Direct localization by cryo-electron microscopy of secondary structural elements in Escherichia coli 23 S rRNA which differ from the corresponding regions in Haloarcula marismortui
Autore:
Matadeen, R; Sergiev, P; Leonov, A; Pape, T; van der Sluis, E; Mueller, F; Osswald, M; von Knoblauch, K; Brimacombe, R; Bogdanov, A; van Heel, M; Dontsova, O;
Indirizzi:
Max Planck Inst Mol Genet, D-14195 Berlin, Germany Max Planck Inst Mol Genet Berlin Germany D-14195 D-14195 Berlin, Germany Univ London Imperial Coll Sci Technol & Med, Dept Biochem, London SW7 2AY,England Univ London Imperial Coll Sci Technol & Med London England SW7 2AY gland Moscow State Univ, AN Belozersky Inst Phys Chem Biol, Moscow 119899, Russia Moscow State Univ Moscow Russia 119899 Chem Biol, Moscow 119899, Russia Moscow State Univ, Dept Chem, Moscow 119899, Russia Moscow State Univ Moscow Russia 119899 Dept Chem, Moscow 119899, Russia
Titolo Testata:
JOURNAL OF MOLECULAR BIOLOGY
fascicolo: 5, volume: 307, anno: 2001,
pagine: 1341 - 1349
SICI:
0022-2836(20010413)307:5<1341:DLBCMO>2.0.ZU;2-C
Fonte:
ISI
Lingua:
ENG
Soggetto:
ELONGATION-FACTOR-G; 16-S RIBOSOMAL-RNA; ANGSTROM RESOLUTION; ANGULAR RECONSTITUTION; 70S RIBOSOME; SUBUNIT; RECONSTRUCTION; VISUALIZATION; TRANSLOCATION;
Keywords:
site-directed mutagenesis; cryo-electron microscopy; X-ray crystallography; 3D structure of rRNA; phylogenetic variation;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
27
Recensione:
Indirizzi per estratti:
Indirizzo: Brimacombe, R Max Planck Inst Mol Genet, Ihnestr 73, D-14195 Berlin, Germany Max Planck Inst Mol Genet Ihnestr 73 Berlin Germany D-14195
Citazione:
R. Matadeen et al., "Direct localization by cryo-electron microscopy of secondary structural elements in Escherichia coli 23 S rRNA which differ from the corresponding regions in Haloarcula marismortui", J MOL BIOL, 307(5), 2001, pp. 1341-1349

Abstract

Insertions were introduced by a two-step mutagenesis procedure into each of five double-helical regions of Escherichia coli 23 S rRNA, so as to extend the helix concerned by 17 bp. The helices chosen were at sites within the23 S molecule (h9, h25, h45, h63 and h98) where significant length variations between different species are known to occur. At each of these positions, with the exception of h45, there are also significant differences between the 23 S rRNAs of E. coli and Haloarcula marismortui. Plasmids carrying the insertions were introduced into an E, coli strain lacking all seven rm operons. In four of the five cases the cells were viable and 50 S subunits could be isolated; only the insertion in h63 was lethal. The modified subunits were examined by cryo-electron microscopy (cryo-EM), with a view to locating extra electron density corresponding to the insertion elements. The results were compared both with the recently determined atomic structure of H. marismortui 23 S rRNA in the 50 S subunit, and with previous 23 S rRNA modelling studies based on cryo-EM reconstructions of E. coli ribosomes. The insertion element in h45 was located by cryo-EM at a position correspondingprecisely to that of the equivalent helix in H. marismortui. The insertionin h98 (which is entirely absent in H. marismortui) was similarly located at a position corresponding precisely to that predicted from the E, coli modelling studies. In the region of h9, the difference between the E, coli and H. marismortui secondary structures is ambiguous, and the extra electron density corresponding to the insertion was seen at a location intermediate between the position of the nearest helix in the atomic structure and that in the modelled structure. Ln the case of h25 (which is about 50 nucleotides longer in H. marismortui), no clear extra cryo-EM density corresponding to the insertion could be observed. (C) 2001 Academic Press.

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Documento generato il 03/04/20 alle ore 11:03:51