The signal recognition particle (SRP) targets nascent proteins to cellular membranes for insertion or secretion by recognizing polypeptides containing an N-terminal signal sequence as they emerge from the ribosome. GTP-dependent binding of SRP to its receptor protein leads to controlled release of the nascent chain into a membrane-spanning translocon pore. Here we show that the association of the SRP with its receptor triggers a marked conformational change in the complex, localizing the SRP RNA and the adjacent signal peptide-binding site at the SRP-receptor heterodimer interface. The orientation of the RNA suggests how peptide binding and GTP hydrolysis can be coupled through direct structural contact during cycles of SRP-directed protein translocation.
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.
The RNA-dependent protein kinase (PKR) is regulated by the binding of double-stranded RNA (dsRNA) or single-stranded RNAs with extensive duplex secondary structure. PKR has an RNA binding domain (RBD) composed of two copies of the dsRNA binding motif (dsRBM). The dsRBM is an alpha-beta-beta-beta-alpha structure present in a number of proteins that bind RNA, and the selectivity demonstrated by these proteins is currently not well understood. We have used affinity cleavage to study the binding of PKR's RBD to RNA. In this study, we site-specifically modified the first dsRBM of PKR's RBD at two different amino acid positions with the hydroxyl radical generator EDTA.Fe. Cleavage by these proteins of a synthetic stem-loop ligand of PKR indicates that PKR's dsRBMI binds the RNA in a preferred orientation, placing the loop between strands beta1 and beta2 near the single-stranded RNA loop. Additional cleavage experiments demonstrated that defects in the RNA stem, such as an A bulge and two GA mismatches, do not dictate dsRBMI's binding orientation preference. Cleavage of VA(I) RNA, an adenoviral RNA inhibitor of PKR, indicates that dsRBMI is bound near the loop of the apical stem of this RNA in the same orientation as observed with the synthetic stem-loop RNA ligands. This work, along with an NMR study of the binding of a dsRBM derived from the Drosophila protein Staufen, indicates that dsRBMs can bind stem-loop RNAs in distinct ways. In addition, the successful application of the affinity cleavage technique to localizing dsRBMI of PKR on stem-loop RNAs and defining its orientation suggests this approach could be applied to dsRBMs found in other proteins.
The RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phosphorylates and inhibits the function of the translation initiation factor eIF-2. PKR has a double-stranded RNA-binding domain (dsRBD) composed of two copies of the dsRNA binding motif (dsRBM). PKR's dsRBD is involved in the regulation of the enzyme as dsRNAs of cellular and viral origins bind to the dsRBD, leading to either activation or inhibition of PKR's kinase activity. In this study, we site-specifically modified each of the dsRBMs of PKR's dsRBD with the hydroxyl radical generator EDTA small middle dotFe and performed cleavage studies on kinase-activating and kinase-inhibiting RNAs. These experiments led to the identification of binding sites for the individual dsRBMs on various RNA ligands including a viral activating RNA (TAR from HIV-1), a viral inhibiting RNA (VA(I) RNA from adenovirus), an aptamer RNA that activates PKR, and a small synthetic inhibiting RNA. These results indicate that some RNAs interact only with one dsRBM, while others can bind both dsRBMs of PKR. In addition, EDTA small middle dotFe modification coupled with site-directed mutagenesis was used to assess the extent of cooperativity in the binding of the two dsRBMs. These experiments support the hypothesis that simultaneous binding of both dsRBMs of PKR occurs on kinase activating RNA ligands.
The RNA-dependent protein kinase (PKR) is an interferoninduced, RNA-activated enzyme that phosphorylates the eukaryotic initiation factor 2α, rendering the translation machinery inactive. Viruses have developed strategies for preventing the action of PKR, one of which is the production of small RNAs that inhibit the enzyme. Epstein-Barr virus (EBV) encodes EBER1, a 167 nucleotide non-coding RNA that is constitutively expressed by the EBV-infected cells. EBER1 binds PKR in vitro and has been shown to prevent inhibition of translation by PKR in vitro. We used affinity cleavage by the EDTA·Fe-modified double-stranded RNA-binding domain (dsRBD) of PKR to show that stem-loop IV (nucleotides 87-123) of EBER1 makes specific contacts with the dsRBD. To further demonstrate the specificity of this interaction, we generated a deletion mutant of EBER1, comprising only stem-loop IV (mEBER1). Cleavage patterns produced on mEBER1 by the bound dsRBD were remarkably similar to those found on full-length EBER1. Using cleavage data from two different dsRBD mutants, we present a model of the interaction of PKR dsRBD and mEBER1.
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