No abstract

The La-related proteins (LARPs) form a diverse group of RNA-binding proteins characterized by the possession of a composite RNA binding unit, the La module. The La module comprises two domains, the La motif (LaM) and the RRM1, which together recognize and bind to a wide array of RNA substrates. Structural information regarding the La module is at present restricted to the prototypic La protein, which acts as an RNA chaperone binding to 3′ UUUOH sequences of nascent RNA polymerase III transcripts. In contrast, LARP6 is implicated in the regulation of collagen synthesis and interacts with a specific stem-loop within the 5′ UTR of the collagen mRNA. Here, we present the structure of the LaM and RRM1 of human LARP6 uncovering in both cases considerable structural variation in comparison to the equivalent domains in La and revealing an unprecedented fold for the RRM1. A mutagenic study guided by the structures revealed that RNA recognition requires synergy between the LaM and RRM1 as well as the participation of the interdomain linker, probably in realizing tandem domain configurations and dynamics required for substrate selectivity. Our study highlights a considerable complexity and plasticity in the architecture of the La module within LARPs.

Rpp20 and Rpp25 are two key subunits of the human endoribonucleases RNase P and MRP. Formation of an Rpp20–Rpp25 complex is critical for enzyme function and sub-cellular localization. We present the first detailed in vitro analysis of their conformational properties, and a biochemical and biophysical characterization of their mutual interaction and RNA recognition. This study specifically examines the role of the Rpp20/Rpp25 association in the formation of the ribonucleoprotein complex. The interaction of the individual subunits with the P3 arm of the RNase MRP RNA is revealed to be negligible whereas the 1:1 Rpp20:Rpp25 complex binds to the same target with an affinity of the order of nM. These results unambiguously demonstrate that Rpp20 and Rpp25 interact with the P3 RNA as a heterodimer, which is formed prior to RNA binding. This creates a platform for the design of future experiments aimed at a better understanding of the function and organization of RNase P and MRP. Finally, analyses of interactions with deletion mutant proteins constructed with successively shorter N- and C-terminal sequences indicate that the Alba-type core domain of both Rpp20 and Rpp25 contains most of the determinants for mutual association and P3 RNA recognition.

S U M M A R YMutants of Pseudomonas aeruginosa 8602 were isolated which, unlike the wild type, were able to grow with butyramide as a carbon source. Six mutants derived from the constitutive strain c 1 1 were shown to produce an enzyme (B amidase) with altered electrophoretic mobility and altered substrate specificity. The apparent K," for butyramide of the B amidase was about a tenth of that of the A amidase and the V,,,,,, was about ten-fold greater. A further mutation produced mutants able to grow on valeramide. I N T R O D U C T I O NPseudomonas aeruginosa 8602 grows in a minimal medium containing either acetamide or propionamide as a carbon + nitrogen source, and both these amides induce the synthesis of an aliphatic amidase (acylamide amidohydrolase EC 3.4.14). This organism is unable to utilize butyramide for growth and Kelly & Clarke (1962) found that acetamide-grown bacteria (and extracts prepared from them) had very little amidase activity with butyramide as a substrate and that butyramide did not induce amidase synthesis in a succinate + minimal salt medium. The magno-constitutive mutant c 1 1 (Brammar, Clarke & Skinner, 1967) is also unable to grow on butyramide and produces an enzyme which is indistinguishable from the wild-type amidase.We have now obtained mutants of strain c 11 which grow on agar plates and in liquid medium with butyramide as the carbon source. The amidase produced by one of these butyramide-utilizing mutants has been purified and compared with that produced by strain c 11. M E T H O D SOrganisms. The parent strain was Pseudomonas aeruginosa 8602. Strain c 11 was a spontaneous mutant, constitutive for amidase (Brammar et al. 1967) and was used to isolate the butyramide-utilizing mutants designated B I to B 6 (see later for genetic descriptions) .Media. The minimal salt medium described by Brammar & Clarke (1964) was used for all growth experiments; solid media contained 1 . 2 % (w/v) Oxoid No. 3 agar. Lactate and pyruvate media were prepared by adding sterile solutions of the sodium or potassium salts aseptically to autoclaved minimal salt medium. Butyramide plates contained 0 . 1 % (w/v) and valeramide plates 0.2y0 (w/v) of the specific amide, and were prepared by adding sterile solutions of the amides to minimal agar. Other media were prepared as described by Brammar et al. (1967). 18-2