Bernard Ehresmann

The fragile X mental retardation protein binds specifically to its mRNA via a purine quartet motif

Fragile X syndrome is caused by the absence of protein FMRP, the function of which is still poorly understood. Previous studies have suggested that FMRP may be involved in various aspects of mRNA metabolism, including transport, stability and/or translatability. FMRP was shown to interact with a subset of brain mRNAs as well as with its own mRNA; however, no specific RNA‐binding site could be identified precisely. Here, we report the identification and characterization of a specific and high affinity binding site for FMRP in the RGG‐coding region of its own mRNA. This site contains a purine quartet motif that is essential for FMRP binding and can be substituted by a heterologous quartet‐forming motif. The specific binding of FMRP to its target site was confirmed further in a reticulocyte lysate through its ability to repress translation of a reporter gene harboring the RNA target site in the 5′‐untranslated region. Our data address interesting questions concerning the role of FMRP in the post‐transcriptional control of its own gene and possibly other target genes.

Staphylococcus aureus RNAIII and the endoribonuclease III coordinately regulate spa gene expression

Staphylococcus aureus RNAIII is one of the largest regulatory RNAs, which controls several virulence genes encoding exoproteins and cell‐wall‐associated proteins. One of the RNAIII effects is the repression of spa gene (coding for the surface protein A) expression. Here, we show that spa repression occurs not only at the transcriptional level but also by RNAIII‐mediated inhibition of translation and degradation of the stable spa mRNA by the double‐strand‐specific endoribonuclease III (RNase III). The 3′ end domain of RNAIII, partially complementary to the 5′ part of spa mRNA, efficiently anneals to spa mRNA through an initial loop–loop interaction. Although this annealing is sufficient to inhibit in vitro the formation of the translation initiation complex, the coordinated action of RNase III is essential in vivo to degrade the mRNA and irreversibly arrest translation. Our results further suggest that RNase III is recruited for targeting the paired RNAs. These findings add further complexity to the expression of the S. aureus virulon.

The structure of threonyl-tRNA synthetase-tRNA(Thr) complex enlightens its repressor activity and reveals an essential zinc ion in the active site

E. coli threonyl-tRNA synthetase (ThrRS) is a class II enzyme that represses the translation of its own mRNA. We report the crystal structure at 2.9 A resolution of the complex between tRNA(Thr) and ThrRS, whose structural features reveal novel strategies for providing specificity in tRNA selection. These include an amino-terminal domain containing a novel protein fold that makes minor groove contacts with the tRNA acceptor stem. The enzyme induces a large deformation of the anticodon loop, resulting in an interaction between two adjacent anticodon bases, which accounts for their prominent role in tRNA identity and translational regulation. A zinc ion found in the active site is implicated in amino acid recognition/discrimination.

The role of exportin‐t in selective nuclear export of mature tRNAs

Exportin‐t (Xpo‐t) is a vertebrate nuclear export receptor for tRNAs that binds tRNA cooperatively with GTP‐loaded Ran. Xpo‐t antibodies are shown to efficiently block tRNA export from Xenopus oocyte nuclei suggesting that it is responsible for at least the majority of tRNA export in these cells. We examine the mechanism by which Xpo‐t–RanGTP specifically exports mature tRNAs rather than other forms of nuclear RNA, including tRNA precursors. Chemical and enzymatic footprinting together with phosphate modification interference reveals an extensive interaction between the backbone of the TΨC and acceptor arms of tRNAPhe and Xpo‐t–RanGTP. Analysis of mutant or precursor tRNA forms demonstrates that, aside from these recognition elements, accurate 5′ and 3′ end‐processing of tRNA affects Xpo‐t–RanGTP interaction and nuclear export, while aminoacylation is not essential. Intron‐containing, end‐processed, pre‐tRNAs can be bound by Xpo‐t–RanGTP and are rapidly exported from the nucleus if Xpo‐t is present in excess. These results suggest that at least two mechanisms are involved in discrimination of pre‐tRNAs and mature tRNAs prior to nuclear export.

Translational control of ribosomal protein S15

The expression of ribosomal protein S15 is shown to be translationally and negatively autocontrolled using a fusion within a reporter gene. Isolation and characterization of several deregulated mutants indicate that the regulatory site (the translational operator site) overlaps the ribosome loading site of the S15 messenger. In this region, three domains, each exhibiting a stem-loop structure, were determined using chemical and enzymatic probes. The most downstream hairpin carries the Shine-Dalgarno sequence and the initiation codon. Genetic and structural data derived from mutants constructed by site-directed mutagenesis show that the operator is a dynamic structure, two domains of which can form a pseudoknot. Binding of S15 to these two domains suggests that the pseudoknot could be stabilized by S15. A model is presented in which two alternative structures would explain the molecular basis of the S15 autocontrol.

Structural basis for the specificity of the initiation of HIV‐1 reverse transcription

Initiation of human immunodeficiency virus type 1 (HIV‐1) reverse transcription requires specific recognition of the viral genome, tRNA3Lys, which acts as primer, and reverse transcriptase (RT). The specificity of this ternary complex is mediated by intricate interactions between HIV‐1 RNA and tRNA3Lys, but remains poorly understood at the three‐dimensional level. We used chemical probing to gain insight into the three‐dimensional structure of the viral RNA–tRNA3Lys complex, and enzymatic footprinting to delineate regions interacting with RT. These and previous experimental data were used to derive a three‐dimensional model of the initiation complex. The viral RNA and tRNA3Lys form a compact structure in which the two RNAs fold into distinct structural domains. The extended interactions between these molecules are not directly recognized by RT. Rather, they favor RT binding by preventing steric clashes between the nucleic acids and the polymerase and inducing a viral RNA–tRNA3Lys conformation which fits perfectly into the nucleic acid binding cleft of RT. Recognition of the 3′ end of tRNA3Lys and of the first template nucleotides by RT is favored by a kink in the template strand promoted by the short junctions present in the previously established secondary structure.

Progression of a loop–loop complex to a four‐way junction is crucial for the activity of a regulatory antisense RNA

The antisense RNA, CopA, regulates the replication frequency of plasmid R1 through inhibition of RepA translation by rapid and specific binding to its target RNA (CopT). The stable CopA–CopT complex is characterized by a four‐way junction structure and a side‐by‐side alignment of two long intramolecular helices. The significance of this structure for binding in vitro and control in vivo was tested by mutations in both CopA and CopT. High rates of stable complex formation in vitro and efficient inhibition in vivo required initial loop–loop complexes to be rapidly converted to extended interactions. These interactions involve asymmetric helix progression and melting of the upper stems of both RNAs to promote the formation of two intermolecular helices. Data presented here delineate the boundaries of these helices and emphasize the need for unimpeded helix propagation. This process is directional, i.e. one of the two intermolecular helices (B) must form first to allow formation of the other (B′). A binding pathway, characterized by a hierarchy of intermediates leading to an irreversible and inhibitory RNA–RNA complex, is proposed.

Computer modeling from solution data of spinach chloroplast and of Xenopus laevis somatic and oocyte 5 S rRNAs

Detailed atomic models of a eubacterial 5 S rRNA (spinach chloroplast 5 S rRNA) and of a eukaryotic 5 S rRNA (somatic and oocyte 5 S rRNA from Xenopus laevis) were built using computer graphic. Both models integrate stereochemical constraints and experimental data on the accessibility of bases and phosphates towards several structure-specific probes. The base sequence was first inserted on to three-dimensional structural fragments picked up in a specially devised databank. The fragments were modified and assembled interactively on an Evans & Sutherland PS330. Modeling was finalized by stereochemical and energy refinement. In spite of some uncertainty in the relative spatial orientation of the substructures, the broad features of the models can be generalized and several conclusions can be reached: (1) both models adopt a distorted Y-shape structure, with helices B and D not far from colinearity; (2) no tertiary interactions exist between loop c and region d or loop e; (3) the internal loops, in particular region d, contain several non-canonical base-pairs of A.A, U.U and A.G types; (4) invariant residues appear to be more important for protein or RNA binding than for maintaining the tertiary structure. The models are corroborated by footprinting experiments with ribosomal proteins and by the analysis of various mutants. Such models help to clarify the structure-function relationship of 5 S rRNA and are useful for designing site-directed mutagenesis experiments.

The sequence of Escherichia coli ribosomal 16 S RNA determined by new rapid gel methods

Ten years ago, we began to determine the sequence of r~bosom~ 16 S RNA from~sc~e~c~~~ coil MRE 600 using the conventionaL sequencing technique of Sanger et al. [ 11. As we pointed out in [2J, a limit in the length of sequence that could be determined was reached, due partly to the intrinsic limitations of the technique itself, and partly to the extreme difficulty of preparing RNA fra~ents from exposed regions which are very susceptible to limited ~bonuclease Tl digestion. Recently a new ribonuclease, extracted from the venom of cobra Nuja oxiana [3], which is specific for double-stranded or base-stacked regions 141 allowed us to prepare new RNA fra~ents different from those previously obtained. Furthermore, the recent development of rapid RNA sequencing techniques, using specific digestions of 5’-32P-labelled RNA fragments and subsequent fractionation of the digests on polyacrylamide gels [5,6], allowed us to make rapid progress. Using the new methodology, we have examined the entire 16 S RNA molecule: the sequences of previously non-ordered regions have been resolved; and several corrections have been made in areas already sequenced using the conventions RNA sequencing technique [lx. We report here the complete sequence of the 16 S RNA encompassing 1542 nucleotides. 2. Materials and methods

Restoration of tRNA-primed()-Strand DNA Synthesis to an HIV-1 Reverse Transcriptase Mutant with Extended tRNAs IMPLICATIONS FOR RETROVIRAL REPLICATION

The mechanism for the initiation of reverse transcription in human immunodeficiency virus type 1 (HIV-1) was studied utilizing a unique reverse transcriptase (RT) mutant altered in its noncatalytic p51 subunit. This mutant (p66/p51Δ13) retains full DNA- and RNA-dependent DNA polymerase activity but has reduced affinity for tRNA, the cognate HIV primer. When the ability to support(-)-strand DNA synthesis on a viral RNA template was evaluated, this mutant initiated from an 18-nucleotide (nt) oligoribo- or oligodeoxyribonucleotide primer complementary to the primer binding site (pbs). However, it failed to do so from natural and synthetic versions of tRNA. tRNA-primed(-)-strand synthesis could, however, be rescued by substituting the 76-nt tRNA with 81- and 107-nt tRNA-DNA chimeras, i.e. tRNA extended by 5 and 31 deoxyribonucleotides complementary to the viral genome upstream of the pbs. These findings imply that through interactions involving its p51 subunit, RT may be required to disrupt additional tRNA-viral RNA duplexes outside the pbs to proceed into productive(-)-strand DNA synthesis. Alternatively, specific interactions between tRNA and HIV-1 RT may be necessary for efficient initiation of(-)-strand DNA synthesis.