The downstream box (DB) has been proposed to enhance translation of several mRNAs and to be a key element controlling the expression of cold-shocked mRNAs. However, the proposal that the DB exerts its effects through a base pairing interaction with the complementary anti-downstream box (antiDB) sequence (nt 1469–1483) located in the penultimate stem (helix 44) of 16S rRNA remains controversial. The existence of this interaction during initiation of protein synthesis under cold-shock conditions has been investigated in the present work using an Escherichia coli strain whose ribosomes lack the potential to base pair with mRNA because of a 12 bp inversion of the antiDB sequence in helix 44. Our results show that this strain is capable of cold acclimation, withstands cold shock, and its ribosomes translate mRNAs that contain or lack DB sequences with similar efficiency, comparable to that of the wild type. The structure of helix 44 in 30S ribosomal subunits from cells grown at 37 °C and from cells subjected to cold shock was also analyzed by binding a 32P-labeled oligonucleotide complementary to the antiDB region and by chemical probing with DMS and kethoxal. Both approaches clearly indicate that this region is in a double-stranded conformation and therefore not available for base pairing with mRNA.

Loop–loop interactions among nucleic acids constitute an important form of molecular recognition in a variety of biological systems. In HIV-1, genomic dimerization involves an intermolecular RNA loop–loop interaction at the dimerization initiation site (DIS), a hairpin located in the 5′ noncoding region that contains an autocomplementary sequence in the loop. Only two major DIS loop sequence variants are observed among natural viral isolates. To investigate sequence and structural constraints on genomic RNA dimerization as well as loop–loop interactions in general, we randomized several or all of the nucleotides in the DIS loop and selected in vitro for dimerization-competent sequences. Surprisingly, increasing interloop complementarity above a threshold of 6 bp did not enhance dimerization, although the combinations of nucleotides forming the theoretically most stable hexanucleotide duplexes were selected. Noncanonical interactions contributed significantly to the stability and/or specificity of the dimeric complexes as demonstrated by the overwhelming bias for noncanonical base pairs closing the loop and covariations between flanking and central loop nucleotides. Degeneration of the entire loop yielded a complex population of dimerization-competent sequences whose consensus sequence resembles that of wild-type HIV-1. We conclude from these findings that the DIS has evolved to satisfy simultaneous constraints for optimal dimerization affinity and the capacity for homodimerization. Furthermore, the most constrained features of the DIS identified by our experiments could be the basis for the rational design of DIS-targeted antiviral compounds.

The antisense RNA CopA binds to the leader region of the repA mRNA (target: CopT). Previous studies on CopA–CopT pairing in vitro showed that the dominant product of antisense RNA–mRNA binding is not a full RNA duplex. We have studied here the structure of CopA–CopT complex, combining chemical and enzymatic probing and computer graphic modeling. CopI, a truncated derivative of CopA unable to bind CopT stably, was also analyzed. We show here that after initial loop–loop interaction (kissing), helix propagation resulted in an extended kissing complex that involves the formation of two intermolecular helices. By introducing mutations (base-pair inversions) into the upper stem regions of CopA and CopT, the boundaries of the two newly formed intermolecular helices were delimited. The resulting extended kissing complex represents a new type of four-way junction structure that adopts an asymmetrical X-shaped conformation formed by two helical domains, each one generated by coaxial stacking of two helices. This structure motif induces a side-by-side alignment of two long intramolecular helices that, in turn, facilitates the formation of an additional intermolecular helix that greatly stabilizes the inhibitory CopA–CopT RNA complex. This stabilizer helix cannot form in CopI–CopT complexes due to absence of the sequences involved. The functional significance of the three-dimensional models of the extended kissing complex (CopI–CopT) and the stable complex (CopA–CopT) are discussed.

Subgenomic (sg) mRNAs are synthesized by (+)-strand RNA viruses to allow for efficient translation of products encoded 3′ in their genomes. This strategy also provides a means for regulating the expression of such products via modulation of sg mRNA accumulation. We have studied the mechanism by which sg mRNAs levels are controlled in tomato bushy stunt virus, a small (+)-strand RNA virus which synthesizes two sg mRNAs during infections. Neither the viral capsid nor movement proteins were found to play any significant role in modulating the accumulation levels of either sg mRNA. Deletion analysis did, however, identify a 12-nt-long RNA sequence located approximately 1,000 nt upstream from the site of initiation of sg mRNA2 synthesis that was required specifically for accumulation of sg mRNA2. Further analysis revealed a potential base-pairing interaction between this sequence and a sequence located just 5′ to the site of initiation for sg mRNA2 synthesis. Mutant genomes in which this interaction was either disrupted or maintained were analyzed and the results indicated a positive correlation between the predicted stability of the base-pairing interaction and the efficiency of sg mRNA2 accumulation. The functional significance of the long-distance interaction was further supported by phylogenetic sequence analysis which revealed conservation of base-pairing interactions of similar stability and relative position in the genomes of different tombusviruses. It is proposed that the upstream sequence represents a cis-acting RNA element which facilitates sg mRNA accumulation by promoting efficient synthesis of sg mRNA2 via a long-distance RNA–RNA interaction.

Histone RNA 3′ processing in vitro produces one or more 5′ cleavage products corresponding to the mature histone mRNA 3′ end, and a group of 3′ cleavage products whose 5′ ends are mostly located several nucleotides downstream of the mRNA 3′ end. The formation of these 3′ products is coupled to the formation of 5′ products and dependent on the U7 snRNP and a heat-labile processing factor. These short 3′ products therefore are a true and general feature of the processing reaction. Identical 3′ products are also formed from a model RNA containing all spacer nucleotides downstream of the mature mRNA 3′ end, but no sequences from the mature mRNA. Again, this reaction is dependent on both the U7 snRNP and a heat-labile factor. Unlike the processing with a full-length histone pre-mRNA, this reaction produces only 3′ but no 5′ fragments. In addition, product formation is inhibited by addition of cap structures at the model RNA 5′ end, indicating that product formation occurs by 5′-3′ exonucleolytic degradation. This degradation of a model 3′ product by a 5′-3′ exonuclease suggests a mechanism for the release of the U7 snRNP after processing by shortening the cut-off histone spacer sequences base paired to U7 RNA.