A method is described to determine simultaneously the effect of any changes in the ribosome-binding site (RBS) of mRNA on translational efficiency in Bacillus subtilis and Escherichia coli in vivo. The approach was used to analyse systematically the influence of spacing between the Shine-Dalgarno sequence and the initiation codon, the three different initiation codons, and RBS secondary structure on translational yields in the two organisms. Both B. subtilis and E. coli exhibited similar spacing optima of 7-9 nucleotides. However, B. subtilis translated messages with spacings shorter than optimal much less efficiently than E. coli. In both organisms, AUG was the preferred initiation codon by two- to threefold. In E. coli GUG was slightly better than UUG while in B. subtilis UUG was better than GUG. The degree of emphasis placed on initiation codon type, as measured by translational yield, was dependent on the strength of the Shine-Dalgarno interaction in both organisms. B. subtilis was also much less able to tolerate secondary structure in the RBS than E. coli. While significant differences were found between the two organisms in the effect of specific RBS elements on translation, other mRNA components in addition to those elements tested appear to be responsible, in part, for translational species specificity. The approach described provides a rapid and systematic means of elucidating such additional determinants.

The influence of ribosome-binding-site elements on translational efficiency in Bacillus subtilis and Escherichia coli in vivo.

RNA molecules face difficulties when folding into their native structures. In the cell, proteins can assist RNAs in reaching their functionally active states by binding and stabilizing a specific structure or, in a quite opposite way, by interacting in a non-specific manner. These proteins can either facilitate RNA-RNA interactions in a reaction termed RNA annealing, or they can resolve non-functional inhibitory structures. The latter is defined as "RNA chaperone activity" and is the main topic of this review. Here we define RNA chaperone activity in a stringent way and we review those proteins for which RNA chaperone activity has been clearly demonstrated. These proteins belong to quite diverse families such as hnRNPs, histone-like proteins, ribosomal proteins, cold shock domain proteins and viral nucleocapsid proteins. DExD/H-box containing RNA helicases are discussed as a special family of enzymes that restructure RNA or RNPs in an ATP-dependent manner. We further address the different mechanisms RNA chaperones might use to promote folding including the recently proposed theory of protein disorder as a key element in triggering RNA-protein interactions. Finally, we present a new website for proteins with RNA chaperone activity which compiles all the information on these proteins with the perspective to promote the understanding of their activity.

RNA chaperones, RNA annealers and RNA helicases.

Publisher Summary This chapter explains and interrelate the results of studies on the structure and functions of S1. This chapter discusses various structure such as amino-acid sequences, four repeating homologous stretches, and secondary structure elements. Several methods have been developed to gain information on the relative positions of individual ribosomal components in situ and thus to obtain the molecular topography of the ribosomal particle. These methods include: (a) cross-linking of neighboring components by means of chemical cross-linking agents or by photoactivation; (b) immunoelectron microscopy— that is, visualization of a component on the image of the ribosome by the means of its specific antibody; and (c) the neutron scattering procedure using ribosomes containing deuterated components. All these procedures have been applied in the case of S1, but the results have not been fully satisfactory. There are two r-proteins that have clear functional roles in protein biosynthesis: S1 on the small ribosomal subunit and L7/L12 (the dimorphic protein) on the large subunit. Together on the ribosome, they amount to 110,000 daltons or over one-eight of the r-protein mass. They are also the best studied of all r-proteins. The structure, function, and evolution of L7/L12 have been described, and the structure and homology relationships of a chloroplast L12 protein are discussed in this chapter.

Structure and functions of ribosomal protein S1.

Ribosomal protein S1 is required for translation of most, if not all, natural mRNAs in Escherichia coli in vivo.

Today every schoolchild learns that DNA makes RNA and RNA makes protein. He is further taught that a messenger RNA molecule is like a string of beads consisting of A, C, G, and U residues. The genetic information is read out (like any written code) in a linear fashion, here in groups of three. Such imagery immediately engenders the notion that RNA functions purely as a one-dimensional structure and that all of its secrets are locked in its sequence of bases. Of course, the latter concept is, in essence, true. Just as the amino acid sequence of a polypeptide chain ultimately determines the three-dimensional folding of a protein, so does the sequence of nucleotides in an RNA molecule dictate its potential for forming internal Watson—Crick base pairs (RNA secondary structure) which may further interact specifically with each other (RNA tertiary structure). Indeed, it has long been accepted that tRNA molecules utilize both base pairing and intramolecular bonds of other types to assume a quite defined shape which allows their specific recognition by ribosomes, aminoacyl-tRNA synthetases, and so on. However, it is a more recent realization that messenger RNAs and ribosomal RNAs can form thermodynamically stable secondary and tertiary structures as well.

Genetic Signals and Nucleotide Sequences in Messenger RNA

Ribosomal 30S protein S1 causes disruption of the secondary structure of certain pyrimidine-containing polynucleotides. Helical poly(U), poly(C, U), and neutral and acidic poly(C) are stoichiometrically converted by S1 to structures indistinguishable from their partially or completely thermally denatured forms, as revealed by circular dichroism. Of the several double- and triple-stranded helical polynucleotides tested that contain one polypurine strand and at least one polypyrimidine strand, only the conformation of the DNA.RNA hybrid, poly(A)-poly(dT), is perturbed. In the presence of S1, this hybrid undergoes a transition to a new structure that has a circular dichroism spectrum unlike either the native or thermally denatured forms. Intercalated ethidium bromide is released from poly(A)-poly(dT) by S1, confirming the occurrence of a conformational rearrangement. The translation inhibitor, autintricarboxylic acid, completely inhibits the action of S1 on polypyrimidines, but has no effect on the conformational perturbation of poly(A(-poly(dT). The possible relation between these observations and the biological function of protein S1 is discussed.

R Ng

Papers

Alteration of polynucleotide secondary structure by ribosomal protein S1