Structural Studies of Ribosomal Proteins

Nikonov, S.V.; Nevskaya, N.; Fedorov, Roman; Khairullina, Alfia R.; Tishchenko, Svetlana; Nikulin, Alexei; Garber, Maria

doi:10.1515/bchm.1998.379.7.795

Cited by 11 publications

(2 citation statements)

References 71 publications

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“…Crystallographic studies of ribosomes proceed at various levels of complexity. Several crystal structures of individual r‐proteins and rRNA fragments have been determined over the past years (for reviews see Moore, 1998; Nikonov et al ., 1998). The next phase will ascertain the relative orientations of the components, and the first complex of an r‐protein with a cognate rRNA fragment has recently been solved (Conn et al ., 1999; Wimberly et al ., 1999).…”

Section: Introductionmentioning

confidence: 99%

Flexibility, conformational diversity and two dimerization modes in complexes of ribosomal protein L12

Wahl

Bourenkov²,

Bartunik³

et al. 2000

EMBO J

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Protein L12, the only multicopy component of the ribosome, is presumed to be involved in the binding of translation factors, stimulating factor-dependent GTP hydrolysis. Crystal structures of L12 from Thermotoga maritima have been solved in two space groups by the multiple anomalous dispersion method and refined at 2.4 and 2.0 Å resolution. In both crystal forms, an asymmetric unit comprises two full-length L12 molecules and two N-terminal L12 fragments that are associated in a specific, hetero-tetrameric complex with one non-crystallographic 2-fold axis. The two fulllength proteins form a tight, symmetric, parallel dimer, mainly through their N-terminal domains. Each monomer of this central dimer additionally associates in a different way with an N-terminal L12 fragment. Both dimerization modes are unlike models proposed previously and suggest that similar complexes may occur in vivo and in situ. The structures also display different L12 monomer conformations, in accord with the suggested dynamic role of the protein in the ribosomal translocation process. The structures have been submitted to the Protein Databank (http://www.rcsb.org/pdb) under accession numbers 1DD3 and 1DD4.

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Section: Introductionmentioning

confidence: 99%

Flexibility, conformational diversity and two dimerization modes in complexes of ribosomal protein L12

Wahl

Bourenkov²,

Bartunik³

et al. 2000

EMBO J

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“…8 and 9). Mapping the rRNA binding sites of ribosomal proteins is a prerequisite for the structural analysis of RNA-protein complexes, which might be as important for the determination of the three-dimensional (3D) structure of the ribosome as the 3D structures of isolated proteins (10,11) and rRNA fragments (12). The latter were fitted into the ribosomal structure and played a prominent role for the interpretation of ribosomal 3D maps determined by cryo-electron microscopy or x-ray analysis (13)(14)(15)(16)(17).…”

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confidence: 99%

Selecting rRNA binding sites for the ribosomal proteins L4 and L6 from randomly fragmented rRNA: Application of a method called SERF

Stelzl

Spahn

Nierhaus

2000

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

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Two-thirds of the 54 proteins of the Escherichia coli ribosome interact directly with the rRNAs, but the rRNA binding sites of only a very few proteins are known. We present a method (selection of random RNA fragments; SERF) that can identify the minimal binding region for proteins within ribonucleo-protein complexes such as the ribosome. The power of the method is exemplified with the ribosomal proteins L4 and L6. Binding sequences are identified for both proteins and characterized by phosphorothioate footprinting. Surprisingly, the binding region of L4, a 53-nt rRNA fragment of domain I of 23S rRNA, can simultaneously and independently bind L24, one of the two assembly initiator proteins of the large subunit. Identifying the minimal length of RNA sequences that bind specifically to a protein is a challenge for structural research. The classical approach of digesting an RNA-protein complex with RNase usually does not give the minimal binding region, because the protein(s) within the complex might hinder the access of an RNase. Alternatively, the RNase can cut the RNA within the complex into short sequences that lose the binding capacity. Crosslinking approaches and protection experiments with base modifying reagents indicate vicinity but not necessarily the binding sequence. We decided to exploit the enormous power of in vitro selection methods that can identify the best fitting RNA sequences from about 10 15 variants (SELEX, systematic evolution of ligands by exponential enrichment; ref. 1). However, when randomized sequences are used as starting conditions, affinity selection methods usually do not lead to the naturally occurring binding site for a target as such.Short random fragments from genomic DNA were used to select the DNA binding site of transcription factor IIIA in vitro (2), and a similar idea has been put forward for the selection of protein-nucleic acid interactions (3). In vivo small DNA fragments randomly obtained from 14 genes were identified that stimulate expression of the lacZ gene (4). Expression libraries made of short rRNA sequences have been used to create resistance against antibiotics in vivo (5, 6).The basic idea presented here is that the use of a pool of random fragments from large RNAs directly reveals the native binding site when selected for affinity to a certain protein in vitro. The SELEX variant is abbreviated SERF for selection of random RNA fragments. The principle is shown in Fig. 1. A pool of random rDNA fragments is generated by a random cutting of rDNA. The rDNA fragments are transcribed into RNA in a second step. The advantages of the SERF method are: (i) the selection does not work on just any RNA-protein interactions but rather on the native ones; (ii) the size of the selected rRNA can be chosen and a series of overlapping fragments can reveal the minimal binding site; and (iii) the selection should be fast and efficient because the variability of the pool is low compared with the starting pool used in classical affinity selection experiments.More than two-thirds of t...

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[18] RNA-protein interactions in ribosomes: In vitro selection from randomly fragmented rRNA

Stelzl

Spahn²,

Nierhaus³

2000

Methods in Enzymology

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Structural Studies of Ribosomal Proteins

Cited by 11 publications

References 71 publications

Flexibility, conformational diversity and two dimerization modes in complexes of ribosomal protein L12

Flexibility, conformational diversity and two dimerization modes in complexes of ribosomal protein L12

Selecting rRNA binding sites for the ribosomal proteins L4 and L6 from randomly fragmented rRNA: Application of a method called SERF

[18] RNA-protein interactions in ribosomes: In vitro selection from randomly fragmented rRNA

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