The two translational release factors of prokaryotes, RF1 and RF2, catalyse the termination of polypeptide synthesis at UAG/UAA and UGA/UAA stop codons, respectively. However, how these polypeptide release factors read both non-identical and identical stop codons is puzzling. Here we describe the basis of this recognition. Swaps of each of the conserved domains between RF1 and RF2 in an RF1-RF2 hybrid led to the identification of a domain that could switch recognition specificity. A genetic selection among clones encoding random variants of this domain showed that the tripeptides Pro-Ala-Thr and Ser-Pro-Phe determine release-factor specificity in vivo in RF1 and RF2, respectively. An in vitro release study of tripeptide variants indicated that the first and third amino acids independently discriminate the second and third purine bases, respectively. Analysis with stop codons containing base analogues indicated that the C2 amino group of purine may be the primary target of discrimination of G from A. These findings show that the discriminator tripeptide of bacterial release factors is functionally equivalent to that of the anticodon of transfer RNA, irrespective of the difference between protein and RNA.
Eukaryotic translation termination is mediated by two interacting release factors, eRF1 and eRF3, which act cooperatively to ensure efficient stop codon recognition and fast polypeptide release. The crystal structures of human and Schizosaccharomyces pombe full-length eRF1 in complex with eRF3 lacking the GTPase domain revealed details of the interaction between these two factors and marked conformational changes in eRF1 that occur upon binding to eRF3, leading eRF1 to resemble a tRNA molecule. Small-angle X-ray scattering analysis of the eRF1/eRF3/GTP complex suggested that eRF1's M domain contacts eRF3's GTPase domain. Consistently, mutation of Arg192, which is predicted to come in close contact with the switch regions of eRF3, revealed its important role for eRF1's stimulatory effect on eRF3's GTPase activity. An ATP molecule used as a crystallization additive was bound in eRF1's putative decoding area. Mutational analysis of the ATP-binding site shed light on the mechanism of stop codon recognition by eRF1.[Keywords: eRF1; eRF3; protein biosynthesis; stop codon recognition; translation termination; translational GTPase] Supplemental material is available at http://www.genesdev.org.
MALAT-1 noncoding RNA is localized to nuclear speckles despite its mRNA-like characteristics. Here, we report the identification of several key factors that promote the localization of MALAT-1 to nuclear speckles and also provide evidence that MALAT-1 is involved in the regulation of gene expression. Heterokaryon assays revealed that MALAT-1 does not shuttle between the nucleus and cytoplasm. RNAi-mediated repression of the nuclear speckle proteins, RNPS1, SRm160, or IBP160, which are well-known mRNA processing factors, resulted in the diffusion of MALAT-1 to the nucleoplasm. We demonstrated that MALAT-1 contains two distinct elements directing transcripts to nuclear speckles, which were also capable of binding to RNPS1 in vitro. Depletion of MALAT-1 represses the expression of several genes. Taken together, our results suggest that RNPS1, SRm160, and IBP160 contribute to the localization of MALAT-1 to nuclear speckles, where MALAT-1 could be involved in regulating gene expression.
] i rises are a pivotal signal for egg activation and are responsible for early embryogenesis (2, 3). Accumulated evidence suggests that Ca 2ϩ oscillations are induced by cytosolic sperm factor introduced into the ooplasm upon sperm-egg fusion (2, 4). Therefore, the identification of the Ca 2ϩ oscillation-inducing sperm factor, which is the egg-activating sperm factor, is currently being studied as a central subject to elucidate the mechanisms of fertilization. Recently, Saunders et al.(5) have reported a novel type of PLC (the enzyme that produces InsP 3 and diacylglycerol from membrane PtdInsP 2 ), PLC, which is specifically expressed in mammalian sperm. The injection of RNA-encoding PLC into mouse eggs causes Ca 2ϩ oscillations and subsequent early embryonic development by expressed PLC at an estimated level comparable with the content in a single sperm (5). The Ca 2ϩ oscillation-inducing activity of sperm extract (4, 6) is lost when pretreated with an antibody against PLC (5). Thus, PLC is considered a strong candidate of the sperm factor. To assure this possibility, it is primarily necessary to examine whether purified PLC protein induces Ca 2ϩ oscillations in the egg. It has been shown that PLC1 (7), -␥1 (8, 9), -␥2 (8), -␦1 (10), and -␦4 (7) are expressed in mammalian sperm and that recombinant PLC1, -␥1, -␥2, and -␦1 failed to cause Ca 2ϩ release in the ooplasm (11). PLC is the smallest PLC identified to date, lacking the N-terminal PH domain (Fig. 1A) (5) that is found in all isoforms of PLC, -␥, and -␦ and is the site for interaction with membrane phospholipids (12). Because PLC as well as PLC␦ lacks a regulatory domain such as the G protein-binding site of PLC or the SH domain of PLC␥ for phosphorylation by tyrosine kinase, the activation mechanism of PLC and PLC␦ is unknown. Therefore, it is also necessary to access how PLC undergoes the active state for production of InsP 3 . Here we first show that recombinant PLC protein induces Ca 2ϩ oscillations in mouse eggs and that PLC possesses an extremely high Ca 2ϩ sensitivity in the PtdInsP 2 hydrolyzing activity to be active even at the resting state of cells. EXPERIMENTAL PROCEDURESCloning of PLC and PLC␦1-cDNA encoding full-length PLC (GenBank TM accession number AF435950) was cloned from a cDNA library originated from mouse testis mRNAs. PLC cDNA was amplified by PCR using Pfu polymerase and the following primers: forward, 5Ј-GAC AAGCGGCCCAGATCATG-3Ј; internal forward primer involving an EcoRI site, 5Ј-GGAATTCATATGGAAAGCCAACTTCATGAG-3Ј; re-* This work was supported by a grant-in-aid for general scientific research (Category B) (to S. M.) from the Japan Ministry of Education, Science, Sports, and Culture. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence (s)
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