Conserved but nonessential interaction of SRP RNA with translation factor EF-G

Sagar, M. Bidya; Lucast, Louise; Doudna, Jennifer A.

doi:10.1261/rna.5266504

Cited by 10 publications

(10 citation statements)

References 38 publications

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“…After air-drying, the filters were quantified on a PhosphorImager. The data were analysed as reported previously 30 ; details of experimental protocols are listed in Supplementary Methods.…”

Section: Double Filter-binding Assaysmentioning

confidence: 99%

Structural basis for 5′-end-specific recognition of guide RNA by the A. fulgidus Piwi protein

Yuan

Meister

et al. 2005

Nature

584

526

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RNA interference (RNAi) is a conserved sequence-specific gene regulatory mechanism 1-3 mediated by the RNA-induced silencing complex (RISC), which is composed of a single-stranded guide RNA and an Argonaute protein. The PIWI domain, a highly conserved motif within Argonaute, has been shown to adopt an RNase H fold 4,5 critical for the endonuclease cleavage activity of RISC 4-6 . Here we report the crystal structure of Archaeoglobus fulgidus Piwi protein bound to double-stranded RNA, thereby identifying the binding pocket for guide-strand 5′-end recognition and providing insight into guide-strand-mediated messenger RNA target recognition. The phosphorylated 5′ end of the guide RNA is anchored within a highly conserved basic pocket, supplemented by the carboxy-terminal carboxylate and a bound divalent cation. The first nucleotide from the 5′ end of the guide RNA is unpaired and stacks over a conserved tyrosine residue, whereas successive nucleotides form a four-base-pair RNA duplex. Mutation of the corresponding amino acids that contact the 5′ phosphate in human Ago2 resulted in attenuated mRNA cleavage activity. Our structure of the Piwi-RNA complex, and that determined elsewhere 7 , provide direct support for the 5′ region of the guide RNA serving as a nucleation site for pairing with target mRNA and for a fixed distance separating the RISC-mediated mRNA cleavage site from the anchored 5′ end of the guide RNA.Small interfering RNAs (siRNAs), the cleavage products of RNase III enzyme Dicer, consist of RNA duplexes that contain two-nucleotide 3′ overhangs and 5′-phosphorylated ends. These unique features at the termini of 19-23-base-pair duplexes, which distinguish siRNAs from other endogenous RNAs, are also required for their functional role in RNAi-mediated processes. Extensive structural and biochemical studies on PAZ (for PIWI/Argonaute/ Zwille)-RNA recognition [8][9][10] , culminating in the structures of PAZ bound to singlestranded RNA 11 and siRNA-like duplexes 12 , have established that the 3′ overhang isCorrespondence and requests for materials should be addressed to D.J.P. (pateld@mskcc.org).. Supplementary Information accompanies the paper on www.nature.com/nature. Competing interests statementThe authors declare that they have no competing financial interests.Coordinates have been deposited in the Protein Data Bank under accession code 1YTU. Here we describe the 2.5-Å crystal structure of the A. fulgidus Piwi protein (Fig. 1a) bound to a 5′-phosphate-containing 21-mer RNA, capable of self-complementary duplex formation (Fig. 1b). The crystallographic asymmetric unit contains two Piwi proteins, each bound to a short four-base-pair segment of duplex, positioned adjacent to the 5′ phosphate and its attached unpaired nucleotide (Fig. 1c). The structure of the A. fulgidus Piwi protein, which consists of a short N-terminal element and two domains labelled A and B (Fig. 1a), is similar (root-mean-square deviation = 0.65Å for Cα) in the free state 5 and in the RNAbound complex. The RNA, whose sequence ...

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Section: Double Filter-binding Assaysmentioning

confidence: 99%

Structural basis for 5′-end-specific recognition of guide RNA by the A. fulgidus Piwi protein

Yuan

Meister

et al. 2005

Nature

584

526

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“…Nonetheless, these results do not address the role of the 4.5S RNA, which also stimulates GTPase activity in the SRP-FtsY complex in vitro and is essential for SRP function in vivo (Brown and Fournier 1984;Peluso et al 2001;Sagar et al 2004;Gu et al 2005). A truncated form of the 114-nucleotide (nt) RNA, including just the distal 44 nt at the hairpin end (domain IV) of the molecule, is sufficient to support cell growth ( Fig.…”

Section: Introductionmentioning

confidence: 97%

SRP RNA provides the physiologically essential GTPase activation function in cotranslational protein targeting

Siu

Spanggord²,

Doudna³

2006

RNA

Self Cite

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The signal recognition particle (SRP) cotranslationally targets proteins to cell membranes by coordinated binding and release of ribosome-associated nascent polypeptides and a membrane-associated SRP receptor. GTP uptake and hydrolysis by the SRPreceptor complex govern this targeting cycle. Because no GTPase-activating proteins (GAPs) are known for the SRP and SRP receptor GTPases, however, it has been unclear whether and how GTP hydrolysis is stimulated during protein trafficking in vivo. Using both biochemical and genetic experiments, we show here that SRP RNA enhances GTPase activity of the SRP-receptor complex above a critical threshold required for cell viability. Furthermore, this stimulation is a property of the SRP RNA tetraloop. SRP RNA tetraloop mutants that confer defective growth phenotypes can assemble into SRP-receptor complexes, but fail to stimulate GTP hydrolysis in these complexes in vitro. Tethered hydroxyl radical probing data reveal that specific positioning of the RNA tetraloop within the SRP-receptor complex is required to stimulate GTPase activity to a level sufficient to support cell growth. These results explain why no external GAP is needed and why the phylogenetically conserved SRP RNA tetraloop is required in vivo.

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“…The SRP RNA has been shown to play an indispensable role in protein targeting both in vitro and in vivo [26][27][28][29][30] . The size of the SRP RNA varies widely from bacteria to yeast and mammalian cells; nevertheless, the most phylogenetically conserved region of the SRP RNA, domain IV, has been maintained in all three kingdoms of life 31,32 .…”

Section: Introductionmentioning

confidence: 99%

Demonstration of a Multistep Mechanism for Assembly of the SRP·SRP Receptor Complex: Implications for the Catalytic Role of SRP RNA

Zhang

Kung

Shan

2008

Journal of Molecular Biology

157

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Two GTPases in the signal recognition particle (SRP) and its receptor (SR) control the delivery of newly synthesized proteins to the ER or plasma membrane. During the protein targeting reaction, the 4.5S SRP RNA accelerates the association between the two GTPases by 400-fold. Using fluorescence resonance energy transfer (FRET), we demonstrate here that formation of a stable SRP•SR complex involves two distinct steps: a fast initial association between SRP and SR to form an early, GTP-independent complex, followed by a GTP-dependent conformational rearrangement to form the stable, final complex. We also found that the 4.5S SRP RNA significantly stabilizes the early, GTP-independent intermediate. Further, mutational analyses show that there is a strong correlation between the ability of the mutant SRP RNAs to stabilize the early intermediate and their ability to accelerate SRP•SR complex formation. We propose that the SRP RNA, by stabilizing the transient early intermediate, can give this intermediate a longer dwell time and therefore a higher probability to rearrange to the final, stable complex. This provides a coherent model that explains how the 4.5S RNA exerts its catalytic role in SRP•SR complex assembly.

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Conserved but nonessential interaction of SRP RNA with translation factor EF-G

Cited by 10 publications

References 38 publications

Structural basis for 5′-end-specific recognition of guide RNA by the A. fulgidus Piwi protein

Structural basis for 5′-end-specific recognition of guide RNA by the A. fulgidus Piwi protein

SRP RNA provides the physiologically essential GTPase activation function in cotranslational protein targeting

Demonstration of a Multistep Mechanism for Assembly of the SRP·SRP Receptor Complex: Implications for the Catalytic Role of SRP RNA

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