Crystallographic and modeling studies of RNase III suggest a mechanism for double-stranded RNA cleavage.

BACKGROUND: Aquifex aeolicus Ribonuclease III (Aa-RNase III) belongs to the family of Mg(2+)-dependent endonucleases that show specificity for double-stranded RNA (dsRNA). RNase III is conserved in all known bacteria and eukaryotes and has 1-2 copies of a 9-residue consensus sequence, known as the RNase III signature motif. The bacterial RNase III proteins are the simplest, consisting of two domains: an N-terminal endonuclease domain, followed by a double-stranded RNA binding domain (dsRBD). The three-dimensional structure of the dsRBD in Escherichia coli RNase III has been elucidated; no structural information is available for the endonuclease domain of any RNase III. RESULTS: We present the crystal structures of the Aa-RNase III endonuclease domain in its ligand-free form and in complex with Mn(2+). The structures reveal a novel protein fold and suggest a mechanism for dsRNA cleavage. On the basis of structural, genetic, and biological data, we have constructed a hypothetical model of Aa-RNase III in complex with dsRNA and Mg(2+) ion, which provides the first glimpse of RNase III in action. CONCLUSIONS: The functional Aa-RNase III dimer is formed via mainly hydrophobic interactions, including a "ball-and-socket" junction that ensures accurate alignment of the two monomers. The fold of the polypeptide chain and its dimerization create a valley with two compound active centers at each end of the valley. The valley can accommodate a dsRNA substrate. Mn(2+) binding has significant impact on crystal packing, intermolecular interactions, thermal stability, and the formation of two RNA-cutting sites within each compound active center.

Biochemical characterization of a cellular structure retaining vegetally localized RNAs in Xenopus late stage oocytes.

Two pathways operate during Xenopus oogenesis to localize a small number of RNAs to the vegetal cortex. Correct localization of these RNAs is essential to normal development as the proteins they encode are involved in specifying cell type and in patterning the early embryo. Binding these RNAs to the vegetal cortex and thus preserving their localized condition is a critical step, although little is known about how this is achieved. In this study, we have used a biochemical approach to examine the anchoring step. Xlsirts, an abundant localized RNA (locRNA), was selectively enriched in a detergent-insoluble fraction (DIF) prepared from oocytes that had completed the RNA localization process. These putative RNA-anchoring complexes were analyzed by density gradient centrifugation and in RNA-protein binding assays. Cortical Xlsirts and other localized RNAs are specifically found in the heavy region of sucrose gradients and in the pellet, quite different from other cellular RNPs. Four proteins were identified by UV-crosslinking that bound the Xlsirts localization signal in the cortex, but not in the soluble fraction. These are likely members of the anchoring complex and appear to include vera, a characterized Vg1 RNA binding protein. Vera was found to co-sediment with other locRNAs found in the vegetal cortex, suggesting that it is a common component of locRNPs. Finally, we found that locRNPs extracted into the soluble fraction had the same buoyant density as typical ooplasmic RNPs. We propose that locRNAs are organized and anchored in the cortex as typical RNPs.

DEADSouth is a germ plasm specific DEAD-box RNA helicase in Xenopus related to eIF4A.

DEADSouth was selected in a screen for localized RNAs in Xenopus oocytes. In situ hybridization analysis shows that DEADSouth localizes to the vegetal cortex via the mitochondrial cloud early in oogenesis and segregates with germ plasm during early embryogenesis. These results lend further support for the general concept that the role of the early RNA localization pathway in Xenopus is to localize germ cell components (reviewed in King, M.L., Zhou, Y., Bubunenko, M. , 1999. BioEssays 21, 546-557). Further analysis shows that DEADSouth is a germline specific RNA, found exclusively within the germ plasm of oocytes and PGCs, as well as in male germ cells. Sequence comparisons with DEADSouth show it to be a member of a small sub-family of the DEAD-box RNA-dependent helicases related to eIF4A.

Xcat2 RNA is a translationally sequestered germ plasm component in Xenopus.

In Xenopus, the inheritance of germ plasm by a small subset of blastomeres during early development is thought to direct these cells into the germ cell lineage. We show that Xcat2 RNA, related to Drosophila nanos, is a germ plasm component that is translationally repressed during oogenesis. Xcat2 protein was not detected in oocytes at times prior to, or after its RNA was localized in germ plasm, suggesting Xcat2 RNA is functionally sequestered soon after transcription. Indeed, Xcat2 RNA is found in a dense non-polysomal compartment in oocytes. Repression of translation was not relieved by substituting the Xcat2 3'UTR with that of beta-globin. Immunodetection of Xcat2 protein during blastula and gastrula stages coincides with the time of symmetric segregation of the germ plasm and a net increase in the number of primordial germ cells. Xcat2 is capable of binding RNA in vitro and we propose that it may function to translationally regulate other RNAs specific to primordial germ cells.

Polarizing genetic information in the egg: RNA localization in the frog oocyte.

RNA localization is a powerful strategy used by cells to localize proteins to subcellular domains and to control protein synthesis regionally. In germ cells, RNA targeting has profound implications for development, setting up polarities in genetic information that drive cell fate during embryogenesis. The frog oocyte offers a useful system for studying the mechanism of RNA localization. Here, we discuss critically the process of RNA localization during frog oogenesis. Three major pathways have been identified that are temporally and spatially separated in oogenesis. Each pathway uses a different mechanism to effect RNA localization. In some cases, localization elements within the 3' untranslated region have been identified and have provided unique insights into the localization process. This important field is still in its infancy, however, and much remains to be learned.

Recognition of novel and divergent higher plant chloroplast ribosomal proteins by Escherichia coli ribosome during in vivo assembly.

Architecture of higher plant chloroplast ribosomes involves additional protein domains over that found in the Escherichia coli ribosome, although the rRNAs in these two kinds of ribosomes are very similar in length and sequence (Subramanian, A. R. (1993) Trends Biochem. Sci. 18, 177-180). Here, we show that two chloroplast-specific protein domains (a novel chloroplast ribosomal protein of the 30 S subunit, called Psrp-1 or S22, and a divergent protein of the 50 S subunit with long terminal extensions and low homology to its E. coli counterpart, L21) are both incorporated in E. coli ribosomes and polysomes when their gene constructs are expressed in E. coli. Also, the 67-residue NH2-terminal extension in chloroplast L21 by itself is incorporated. Thus, our results indicate preexisting binding sites for novel chloroplast-specific ribosomal proteins/domains on eubacterial ribosomes. Additionally, we observed cleavage of the chloroplast-targeting transit peptide (present in the expressed Psrp-1 precursor), indicating protease(s) of the required specificity in E. coli cells. The expression of chloroplast L21 with its NH2-terminal extension was inhibitory to E. coli growth, suggesting a drastic effect of the latter on some property of L21. Expression of Psrp-1 was neutral, consistent with a function only in chloroplast translation. Based on analysis of the assembly of Psrp-1 and various L21 fragments in E. coli ribosomes, a general model for studying ribosomal protein-ribosome interactions is suggested.

Protein substitution in chloroplast ribosome evolution. A eukaryotic cytosolic protein has replaced its organelle homologue (L23) in spinach.

The chloroplast translational system differs from the eubacterial ones in containing several ribosomal proteins (RPs) that have no apparent homologues in eubacteria, and in having their RP genes distributed in two cellular genome compartments. The genes maintained in the organelle genome encode mainly ribosome assembly proteins. The discovery in spinach and related plants (Caryophyllidae) of a disrupted chloroplast gene encoding the ribosome assembly protein L23 raised speculations about the transfer of the functional rpl23 gene to the nucleus or the evolutionary loss of L23 protein requirement. To solve this problem, we overexpressed in E. coli the intact rpl23 gene from corn (Zea mays), purified the protein and raised antibodies. Based on immunoanalysis, we show that a prokaryotic-type L23 protein is absent in spinach. Concomittantly we have isolated a new protein from spinach chloroplast 50 S ribosomal subunits and determined its amino acid sequence. The data revealed an unexpectedly high sequence identity to the eukaryotic family of cytosolic L23 proteins (reported from yeast, trypanosome and rat), with conservation of a peptide motif responsible for the specific interaction of these proteins with domain III of 26 S and 23 S rRNA. We propose that the prokaryotic-type L23 protein in the chloroplast ribosomes of Caryophyllidae has been replaced by a homologue of the eukaryotic cytosolic L23 family. These results represent the first case of a protein (gene) substitution in chloroplast ribosome evolution, and open a new view on how the nuclear genome could progressively exert stronger control over the chloroplast translational system. We also describe experiments on the incorporation of chloroplast L23 into E. coli ribosomes, its effect on cell growth, and an unexpected immuno cross-reaction between two chloroplast RP families.

[Study of segments of 23S RNA, important for interaction with elongation factors Tu and G using complementary DNA-oligonucleotides]. / Issledovanie uchastkov 23S RNK, vazhnykh dlia vzaimodeistviia s faktorami élongatsii Tu i G, s pomoshch'iu komplementarnykh DNA-oligonukleotidov.

Several oligonucleotides complementary to different 23S RNA regions were tested in the elongation factor-dependent reactions of the ribosomes. It was found that the 1088-1100 and 1127-1140 sequence parts of the 23S RNA (binding regions for the L11 protein) are very important for EF-G function. The EF-Tu function is markedly less affected by these nucleotides. The probable role of 23S RNA function is discussed.

A novel operon organization involving the genes for chorismate synthase (aromatic biosynthesis pathway) and ribosomal GTPase center proteins (L11, L1, L10, L12: rplKAJL) in cyanobacterium Synechocystis PCC 6803.

Many of the ribosomal protein (RP) genes in both bacterial and chloroplast genomes occur, for reasons not yet understood, in operons that include nonribosomal genes. Here we report such an operon organization in a cyanobacterium (Synechocystis PCC6803) involving the genes for four RPs that are important in the GTPase function of the ribosome and the aroC gene encoding chorismate synthase, a key enzyme in the shikimate pathway for biosynthesis of aromatic amino acids and cell wall components. The Synechocystis aroC encodes a 362-amino-acid residue protein which is 52, 60, and 68% identical to two eubacterial (both 52%), yeast, and a higher plant (Corydalis) chorismate synthase, respectively. The gene was overexpressed in Escherichia coli, and the gene product was shown to cross-react with antibodies to Corydalis chorismate synthase; it also complemented an aroC-lacking E. coli strain. The Synechocystis rpl1 and rpl11 genes encode polypeptides of 237 and 141 amino acid residues, respectively, also with high sequence identities to the corresponding RP sequences from other eubacteria and higher plant chloroplasts. The gene order is shown to be: rpl11-86bp spacer-rpl1-460bp spacer-rpl10-87-bp spacer-rpl12-206bp spacer-aroC. Southern and Northern blot analyses of Synechocystis DNA and RNA, respectively, revealed a single cluster of these genes per genome which is transcribed from a common promoter to an unusually long, approximately 9500-nucleotide transcript. Several constructs of the cyanobacterial aroC and rpl12 genes were made and expressed in E. coli to examine the mechanisms for their very differential expression from a polycistronic mRNA (e.g. four copies L12/ribosome; chorismate synthase, a non-abundant protein). These results present the first biochemical/molecular genetic evidence of shikimate pathway in the cyanobacterial group.

The 3'-terminal untranslated region of alfalfa mosaic virus RNA 4 facilitates the RNA entry into translation in a cell-free system.

In order to understand the role of the 3'-terminal untranslated region (3'-UTR) of alfalfa mosaic virus (AlMV) RNA 4 in viral RNA translation we have constructed the RNA derivatives differing in the length of their 3'-terminal portions and expressed them in a wheat germ extract. The result shows that the removal of the 3'-UTR from AlMV RNA 4 causes a lagged RNA translation in the cell-free system as compared with the translation of the full length RNA 4, thus suggesting the involvement of the 3'-UTR in the translation initiation pathway.

The length of the interdomain region of the L7/L12 protein is important for its function.

Several mutated L7/L12 proteins with changed interdomain regions were obtained. The results showed that the flexible region comprising the 39-52 amino acid residues is functionally important. Its length, but not its amino acid composition, is crucial for the function.

Novel data on interactions of elongation factor Ts.

Interactions of EF-Ts with EF-Tu at all steps of the elongation cycle were studied by limited trypsinolysis, gel-filtration, analytical centrifugation and fluorescence polarization techniques. It is shown that EF-Ts does not dissociate from EF-Tu after GDP to GTP exchange, but remains bound to the Aa-tRNA.EF-Tu.GTP complex up to GTP hydrolysis stage on the ribosome. The possible role of these interactions is discussed.

[Elongation factor EF-Ts interacts with the aminoacyl-tRNA.EF-Tu.GTP complex]. / Faktor élongatsii EF-Ts vzaimodeistvuet s kompleksom aminoatsil-tRNK.EF-Tu.GTP.

The fluorescence polarization technique has been used to study the interaction of the EF-Ts dansyl derivative with EF-Tu after nucleotide exchange and binding of the aminoacyl-tRNA to EF-Tu.GTP. It is shown that the ternary complex formation results in the increase of EF-Ts affinity to EF-Tu and EF-Ts remains bound to EF-Tu up to the GTP hydrolysis stage on the ribosome.

Overexpression of L7/L12 protein with mutations in its flexible region.

Using restriction enzymes and polymerase chain reaction, three mutated forms of L7/L12 proteins with deleted 38-46, 44-52 and 38-52 residues were obtained. These mutant proteins were isolated in a preparative scale and were shown to bind to ribosomes and to affect ribosomal function.

[Ef-Ts elongation factor interacts with elongation factor EF-Tu on ribosomes prior to the GTP hydrolysis stage]. / Faktor élongatsii EF-Ts vzaimodeistvuet s faktorom élongatsii EF-Tu na ribosomakh do stadii gidroliza GTP.

Methods of high-speed centrifugation and limited proteolysis were used to probe the interaction of EF-Tu with EF-Ts on the ribosome. It is shown that EF-Ts dissociates from EF-Tu only after EF-Tu-mediated GTP hydrolysis, i.e. EF-Ts within the EF-Tu.ribosome complexes in the pre-GTP-hydrolysis state co-sediments with the ribosomes and its rate of proteolysis is distinct from that of free EF-Ts. Moreover, as seen from the difference in sensitivity to trypsin of ribosomal proteins L19 and L27 EF-Ts affects the interaction of EF-Tu with the ribosome.

Elongation factors Tu and G change their conformation on interaction with ribosomes.

Limited trypsinolysis was used to study conformational changes in elongation factors Tu and G. The trypsin cleavage rates of the factors differed and depended on both their interaction with ligands and the presence or absence of ribosomes. When the factors were bound to ribosomes, changes in their sensitivity to trypsin were observed depending on whether GDP or GTP was present in the complex, i.e. on the hydrolysis state of the guanine nucleotide ligand. The possible significance of factor structural changes for their functioning is discussed.

Conformational changes in ribosomes upon interaction with elongation factors.

Conformational changes in the ribosomes upon interaction with EF-Tu were studied by limited proteolysis with a set of proteases. The main results are: (1) The cleavage rate of S1 protein strongly depends on the cooperative effect of poly(U) and tRNA: (2) The conformation of L7/L12 proteins is modulated by interaction of elongation factors with the ribosome and depends on hydrolysis of GTP; (3) The sensitivity of some ribosomal proteins (S6, S7, S18, S19, L9, L16, L19, and L27) to proteases changes upon binding of EF-Tu and depends on the ribosome functional state in accordance with GTP hydrolysis. Most of these proteins are located far from the factor-binding center of the ribosome. The possible mechanism of conformational changes is discussed.