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- Mulder, Frans, et al.
(författare)
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Conformation and dynamics of ribosomal stalk protein L12 in solution and on the ribosome
- 2004
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 43:20, s. 5930-5936
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Tidskriftsartikel (refereegranskat)abstract
- During translation, the ribosome and several of its constituent proteins undergo structural transitions between different functional states. Protein L12, present in four copies in prokaryotic ribosomes, forms a flexible "stalk" with key functions in factor-dependent GTP hydrolysis during translocation. Here we have used heteronuclear NMR spectroscopy to characterize L12 conformation and dynamics in solution and on the ribosome. Isolated L 12 forms a symmetric dimer mediated by the N-terminal domains (NTDs), to which each C-terminal domain (CTD) is connected via an unstructured hinge segment. The overall structure can be described as three ellipsoids joined by flexible linkers. No persistent contacts are seen between the two CTDs, or between the NTD and CTD in the L12 dimer in solution. In the H-1-N-15 HSQC spectrum of the Escherichia coli 70S ribosome, a single set of cross-peaks are observed for residues 40-120 of L12, the intensities of which correspond to only two of four protein copies. The structure of the CTDs observed on the ribosome is indistinguishable from that of isolated L12. These results indicate that two CTDs with identical average structures are mobile and extend away from the ribosome, while the other two copies most likely interact tightly with the ribosome even in the absence of translational factors.
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| 3. |
- Sanyal, Suparna, et al.
(författare)
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23S rRNA assisted folding of cytoplasmic malate dehydrogenase is distinctly different from its self-folding
- 2002
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 1362-4962. ; 30:11, s. 2390-2397
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Tidskriftsartikel (refereegranskat)abstract
- The role of the 50S particle of Escherichia coli ribosome and its 23S rRNA in the refolding and subunit association of dimeric porcine heart cytoplasmic malate dehydrogenase (s-MDH) has been investigated. The self-reconstitution of s-MDH is governed by two parallel pathways representing the folding of the inactive monomeric and the dimeric intermediates. However, in the presence of these folding modulators, only one first order kinetics was observed. To understand whether this involved the folding of the monomers or the dimers, subunit association of s-MDH was studied using fluorescein-5-isothiocyanate-rhodamine-isothiocyanate (FITC-RITC) fluorescence energy transfer and chemical crosslinking with gluteraldehyde. The observation suggests that during refolding the interaction of the unstructured monomers of s-MDH with these ribosomal folding modulators leads to very fast formation of structured monomers that immediately dimerise. These inactive dimers then fold to the native ones, which is the rate limiting step in 23S or 50S assisted refolding of s-MDH. Furthermore, the sequential action of the two fragments of domain V of 23S rRNA has been investigated in order to elucidate the mechanism. The central loop of domain V of 23S rRNA (RNA1) traps the monomeric intermediates, and when they are released by the upper stem-loop region of the domain V of 23S rRNA (RNA2) they are already structured enough to form dimeric intermediates which are directed towards the proper folding pathway.
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