Interaction of tRNAs with the ribosome at the A and P sites.

Dabrowski, Marylena; Spahn, C.M.T.; Nierhaus, Knud H.

doi:10.1002/j.1460-2075.1995.tb00168.x

Cited by 39 publications

(39 citation statements)

References 21 publications

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“…Statistically, one phosphate group per molecule containing a sulfur atom instead of a nonbridging oxygen is randomly incorporated 5Ј to A, U, G, or C into the RNA by T7 in vitro transcription. The resulting RNA grossly maintains its biological activity as demonstrated in a comprehensive functional analysis with tRNAs (38). Adding the small inert iodine molecule I 2 induces a cleavage at the modified phosphate group, provided that the sulfur modification does not prevent complex formation and that iodine has access within the complex.…”

Section: Resultsmentioning

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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Section: Resultsmentioning

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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“…Transcribed RNA containing phosphorothioates has about the same biological activity as its unmodified counterpart; this could be demonstrated with tRNAs and mRNAs [5][6][7]12]. Here 5S rRNA was used containing six or less phosphorothioate residues per molecule.…”

Section: Resultsmentioning

confidence: 99%

“…This approach was successfully applied in studies concerning interactions of sugarphosphate backbones of tRNAs with aminoacyl-tRNA synthetases [5,6] and of tRNA as well as of mRNA with ribosomes [7,8].…”

Section: Introductionmentioning

confidence: 99%

5S rRNA sugar‐phosphate backbone protection in complexes with specific ribosomal proteins

et al. 1996

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5S ribosomal RNA forms stable specific complexes with ribosomal proteins LI8, L25 and L5. In this work, interaction of phosphate residues of E. coli 5S rRNA within 5S rRNA-protein complexes has been studied. For this purpose 5S rRNA with statistically distributed phosphorothioate residues has been used for complex formation and the accessibility of phosphorothioates to iodine cleavage in the complex and in the free state has been studied. In free 5S rRNA, the phosphate residue at A73 was partially protected, probably due to being involved in the organization of the spatial structure of 5S rRNA. This protection is stronger in the complex with three proteins when the 5S rRNA structure is stabilized. In the 5S rRNA-L18 complex only two phosphate groups, G7 and A34, were protected. L25 in a complex with 5S rRNA protects large numbers of phosphorothioate groups coneentrating in two clusters, indicating the possibility of two binding sites for this protein on 5S rRNA. The protection pattern differs from that for individual proteins because of the possible rearrangement of the structure.

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“…The resulting transcripts were eluted from 10% denaturing polyacrylamide gels, 5Ј-dephosphorylated, and [ 32 P]-labeled by T4 polynucleotide kinase and [␥-32 P]ATP, followed by a second gel purification. Templates for in vitro transcription were pSTtPhe (14) for tRNA Phe (E. coli), and p67YF0 for tRNA Phe (Saccharomyces cerevisiae, yeast) (16); both were linearized with BstNI. For tRNA f Met , a template was generated from a tRNA f Met precursor clone.…”

Section: Generation Of Phosphorothioate-containing Trnas Andmentioning

confidence: 99%

“…In these experiments, the accessibility of phosphorothioates in ribosome-bound tRNAs toward iodine cleavage was monitored (14). Here, by using the substitution-interference analysis outlined below, we show that three Rp-phosphate oxygens in the anticodon loop of tRNA are required for functional mRNA-dependent binding to the ribosomal P site.…”

mentioning

confidence: 96%

Identification of specific Rp-phosphate oxygens in the tRNA anticodon loop required for ribosomal P-site binding

Schnitzer

Ahsen

1997

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

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ABSTRACTtRNA binding to the ribosomal P site is dependent not only on correct codon-anticodon interaction but also involves identification of structural elements of tRNA by the ribosome. By using a phosphorothioate substitutioninterference approach, we identified specific nonbridging Rp-phosphate oxygens in the anticodon loop of tRNA Phe from Escherichia coli which are required for P-site binding. Stereospecific involvement of phosphate oxygens at these positions was confirmed by using synthetic anticodon arm analogues at which single Rp-or Sp-phosphorothioates were incorporated. Identical interference results with yeast tRNA Phe and E. coli tRNA f Met indicate a common backbone conformation or common recognition elements in the anticodon loop of tRNAs. N-ethyl-N-nitrosourea modification-interference experiments with natural tRNAs point to the importance of the same phosphates in the loop. Guided by the crystal structure of tRNA Phe , we propose that specific Rp-phosphate oxygens are required for anticodon loop (''U-turn'') stabilization or are involved in interactions with the ribosome on correct tRNAmRNA complex formation.

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Interaction of tRNAs with the ribosome at the A and P sites.

Cited by 39 publications

References 21 publications

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

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

5S rRNA sugar‐phosphate backbone protection in complexes with specific ribosomal proteins

Identification of specific Rp-phosphate oxygens in the tRNA anticodon loop required for ribosomal P-site binding

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