Probing the relationship between Gram-negative and Gram-positive S1 proteins by sequence analysis

Escherichia coli ribosomal protein S1 is required for the translation initiation of messenger RNAs, in particular when their Shine–Dalgarno sequence is degenerated. Closely related forms of the protein, composed of the same number of domains (six), are found in all Gram-negative bacteria. More distant proteins, generally formed of fewer domains, have been identified, by sequence similarities, in Gram-positive bacteria and are also termed ‘S1 proteins’. However in the absence of functional information, it is generally difficult to ascertain their relationship with Gram-negative S1. In this article, we report the solution structure of the fourth and sixth domains of the E. coli protein S1 and show that it is possible to characterize their β-barrel by a consensus sequence that allows a precise identification of all domains in Gram-negative and Gram-positive S1 proteins. In addition, we show that it is possible to discriminate between five domain types corresponding to the domains 1, 2, 3, 4–5 and 6 of E. coli S1 on the basis of their sequence. This enabled us to identify the nature of the domains present in Gram-positive proteins and, subsequently, to probe the filiations between all forms of S1

Etude fonctionnelle et structurale, par RMN, de la région C-terminale de la protéine ribosomique S1 d'Escherichia coli (caractérisation d'un mécanisme unique de reconnaissance des ARNs)

La protéine SI est la plus grande des protéines du ribosome d' Escherichia coli. Cette protéine modulaire est composée de six répétitions d'un domaine conservé. SI joue un rôle essentiel dans le démarrage de la traduction en permettant l'initiation de la traduction des messagers procaryotes (gram négatif) dont la région Shine-Dalgamo (SD) est dégénérée ou absente. SI est aussi utilisée par plusieurs bactériophages. Entre autre, elle est capable d'accélérer l'activité de l'endoribonucléase RegB du bactériophage T4, dont la fonction est d'inactiver certains messagers du phage en les clivant au milieu de leur séquence SD. Il a été montré que les fonctions de SI, dans la traduction et dans l'activation de RegB, sont portées par la même région (fragment F345) et semblent correspondre au même mécanisme moléculaire. Cependant, son mode d'action reste inconnu. Notre objectif est donc d'étudier par RMN l'organisation de cette région de SI et d'analyser ses interactions avec différents ARN afin de proposer un mécanisme moléculaire. J'ai caractérisé par RMN les surfaces d'interactions entre les domaines 3, 4 et 5 au sein du fragment F345 ce qui m'a permis de proposer un modèle d'organisation de cette région. Les données trouvées dans la littérature associées aux résultats issus des études d'interactions de SI avec différents ARN ont permis de faire l'hypothèse que la fonction de SI ne serait pas simplement de reconnaître une région particulière de l'ARN. SI serait capable de se lier à n'importe quel ARN et d'induire une déformation favorisant l'interaction de la séquence avec un troisième partenaire (le ribosome dans le cadre de la traduction ou encore la ribonucléase RegB).SI protein is the largest ribosomal protein of Escherichia coli. This modular protein is .composed of six identical motifs. SI plays a key role in the translation initiation of prokaryote messengers when the Shine-Dalgamo sequence is degenerated or lacks. SI is used by several phages. This protein is able to accelerate the endoribonuclease RegB activity which function is to inactivate sorne of the phage messengers by cleaving them at the rniddle of their SD sequences. One SI region (F345) presents SI functions of translation and RegB activation, it corresponds to the same molecular mechanism but its role remains unknown. Our aim is to study, by NMR, the organisation of SI region and analyse the interactions with several RNA to suggest a molecular mechanism. 1 characterized, by NMR, interactions surfaces between domains 3, 4 and 5 in the F345 fragment, which allows us to suggest an organisation model of this region. Data found in the bibliography associated to the results of SI interaction studies with different RNA show that SI is able to bind any RNA and to induce a deformation that promotes the interaction with a third partner (Ribosome in the case of translation or RegB ribonuclease).ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Structural and functional studies of RegB, a new member of a family of sequence-specific ribonucleases involved in mRNA inactivation on the ribosome.

International audienceThe RegB endoribonuclease participates in the bacteriophage T4 life-cycle by favoring early messenger RNA breakdown. RegB specifically cleaves GGAG sequences found in intergenic regions, mainly in translation initiation sites. Its activity is very low but can be enhanced up to 100-fold by the ribosomal 30S subunit or by ribosomal protein S1. RegB has no significant sequence homology to any known protein. Here we used NMR to solve the structure of RegB and map its interactions with two RNA substrates. We also generated a collection of mutants affected in RegB function. Our results show that, despite the absence of any sequence homology, RegB has structural similarities with two Escherichia coli ribonucleases involved in mRNA inactivation on translating ribosomes: YoeB and RelE. Although these ribonucleases have different catalytic sites, we propose that RegB is a new member of the RelE/YoeB structural and functional family of ribonucleases specialized in mRNA inactivation within the ribosome

S1 ribosomal protein functions in translation initiation and ribonuclease RegB activation are mediated by similar RNA/protein interactions. An NMR and SAXS analysis.

International audienceThe ribosomal protein S1, in Escherichia coli, is necessary for the recognition by the ribosome of the translation initiation codon of most messenger RNAs. It also participates in other functions. In particular, it stimulates the T4 endoribonuclease RegB, which inactivates some of the phage mRNAs, when their translation is no longer required, by cleaving them in the middle of their Shine- Dalgarno sequence. In each function, S1 seems to target very different RNAs, which led to the hypothesis that it possesses different RNA binding sites. We previously demonstrated that the ability of S1 to activate RegB is carried by a fragment of the protein formed of three consecutive domains (domains D3, D4 and D5). The same fragment plays a central role in all other functions. We analyzed its structural organization and its interactions with three RNAs: two RegB substrates and a translation initiation region. We show that these three RNAs bind the same area of the protein through a set of systematic (common to the three RNAs) and specific (RNA-dependent) interactions. We also show that, in the absence of RNA, the D4 and D5 domains are associated while the D3 and D4 domains are in equilibrium between an open (non-interacting) and a closed (weakly interacting) form and that RNA binding induces a structural reorganization of the fragment. All these results suggest that the ability of S1 to recognize different RNAs results from a high adaptability of both its structure and its binding surface