The Location and the Significance of a Cross-link Between the Sarcin/Ricin Domain of Ribosomal RNA and the Elongation Factor-G

Chan, Yuen‐Ling; Correll, Carl C.; Wool, Ira G.

doi:10.1016/j.jmb.2004.01.020

Cited by 13 publications

(13 citation statements)

References 39 publications

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“…The low efficiency may also be due to the suboptimal orientation of the nucleotide involved in cross-linking. Moreover, RNA and proteins are sometimes cross-linked by UV irradiation with low efficiency (39). The RNA-RNA cross-links were formed at higher efficiencies (5–7%), probably because many uridines are involved in base pairing and are therefore positioned for efficient RNA-RNA cross-linking.…”

Section: Resultsmentioning

confidence: 99%

U30 of 7SK RNA Forms a Specific Photo-cross-link with Hexim1 in the Context of Both a Minimal RNA-binding site and a Fully Reconstituted 7SK/Hexim1/P-TEFb Ribonucleoprotein Complex

Bart

Baigude

Rana

2009

Journal of Molecular Biology

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Eukaryotic transcription by RNA polymerase II is a highly regulated process and mainly divided into three major steps: initiation, elongation, and termination. Each step of transcription is controlled by a number of cellular factors. Positive transcription factor b, P-TEFb, is composed of cyclin-dependent kinase 9 and a regulatory cyclin (T1/T2). P-TEFb promotes transcriptional elongation of RNA polymerase II by using the catalytic function of CDK9 to phosphorylate various substrates during transcription. P-TEFb is inactivated by sequestration in a complex with the Hexim1 protein and 7SK RNA. The structure of this inactive P-TEFb complex and the mechanisms controlling its equilibrium with the active complex are poorly understood. Here, we used a photoactive nucleotide, 4-thioU, to study the interactions between 7SK RNA and Hexim1. We identified a specific cross-link between nucleotide U30 of 7SK RNA and amino acids 210–220 of Hexim1, in the context of both a minimal RNA-binding site and a full reconstituted 7SK/Hexim1/P-TEFb ribonucleoprotein complex. We also show that a minimal 7SK RNA hairpin comprising nucleotides 24–87 can bind specifically to Hexim1 in vivo. Our results directly demonstrate that the Hexim1 binding site is located in the 24–87 region of 7SK RNA and that the protein residues outside the basic domain of Hexim1 are involved in specific RNA interactions.

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Section: Resultsmentioning

confidence: 99%

U30 of 7SK RNA Forms a Specific Photo-cross-link with Hexim1 in the Context of Both a Minimal RNA-binding site and a Fully Reconstituted 7SK/Hexim1/P-TEFb Ribonucleoprotein Complex

Bart

Baigude

Rana

2009

Journal of Molecular Biology

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“…The ribosomal site(s) to which EF-G binds is (are) in the 50 S subunit, 26 and the most critical portion is in the SRL. [1][2][3][4] Efficient binding of EF-G to the ribosome requires GTP; the GTP is hydrolyzed by EF-G and the complex EF-G$GDP is released from the ribosome. However, the EF-G$GDP$50 S subunit complex is stabilized by the antibiotic fusidic acid.…”

Section: A2531 Mutations Decrease Polyphenylalanine Synthesismentioning

confidence: 99%

A Pathway for the Transmission of Allosteric Signals in the Ribosome through a Network of RNA Tertiary Interactions

Chan

Dresios

Wool

2006

Journal of Molecular Biology

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“…14 According to structural data, the SRD is located close to the catalytic center of bound elongation factors. 21 Deletion, mutation and disruption of SRD of the ribosomal Helix 95 are lethal. 22–24 There are several other universally-conserved SR motifs in the ribosome.…”

Section: Introductionmentioning

confidence: 99%

Isosteric and Nonisosteric Base Pairs in RNA Motifs: Molecular Dynamics and Bioinformatics Study of the Sarcin–Ricin Internal Loop

et al. 2013

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The Sarcin-Ricin RNA motif (SR motif) is one of the most prominent recurrent RNA building blocks that occurs in many different RNA contexts and folds autonomously, i.e., in a context-independent manner. In this study, we combined bioinformatics analysis with explicit-solvent molecular dynamics (MD) simulations to better understand the relation between the RNA sequence and the evolutionary patterns of SR motif. SHAPE probing experiment was also performed to confirm fidelity of MD simulations. We identified 57 instances of the SR motif in a non-redundant subset of the RNA X-ray structure database and analyzed their basepairing, base-phosphate, and backbone-backbone interactions. We extracted sequences aligned to these instances from large ribosomal RNA alignments to determine frequency of occurrence for different sequence variants. We then used a simple scoring scheme based on isostericity to suggest 10 sequence variants with highly variable expected degree of compatibility with the SR motif 3D structure. We carried out MD simulations of SR motifs with these base substitutions. Non isosteric base substitutions led to unstable structures, but so did isosteric substitutions which were unable to make key base-phosphate interactions. MD technique explains why some potentially isosteric SR motifs are not realized during evolution. We also found that inability to form stable cWW geometry is an important factor in case of the first base pair of the flexible region of the SR motif. Comparison of structural, bioinformatics, SHAPE probing and MD simulation data reveals that explicit solvent MD simulations neatly reflect viability of different sequence variants of the SR motif. Thus, MD simulations can efficiently complement bioinformatics tools in studies of conservation patterns of RNA motifs and provide atomistic insight into the role of their different signature interactions.

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The Location and the Significance of a Cross-link Between the Sarcin/Ricin Domain of Ribosomal RNA and the Elongation Factor-G

Cited by 13 publications

References 39 publications

U30 of 7SK RNA Forms a Specific Photo-cross-link with Hexim1 in the Context of Both a Minimal RNA-binding site and a Fully Reconstituted 7SK/Hexim1/P-TEFb Ribonucleoprotein Complex

U30 of 7SK RNA Forms a Specific Photo-cross-link with Hexim1 in the Context of Both a Minimal RNA-binding site and a Fully Reconstituted 7SK/Hexim1/P-TEFb Ribonucleoprotein Complex

A Pathway for the Transmission of Allosteric Signals in the Ribosome through a Network of RNA Tertiary Interactions

Isosteric and Nonisosteric Base Pairs in RNA Motifs: Molecular Dynamics and Bioinformatics Study of the Sarcin–Ricin Internal Loop

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