Loop–loop tertiary interactions play a key role in the folding and catalytic activity of natural hammerhead ribozymes. Using a combination of NMR spectroscopy, site-directed mutagenesis and kinetic and infectivity analyses, we have examined the structure and function of loops 1 and 2 of the (+) and (–) hammerheads of chrysanthemum chlorotic mottle viroid RNA. In both hammerheads, loop 1 is a heptanucleotide hairpin loop containing an exposed U at its 5′ side and an extrahelical U at its 3′-side critical for the catalytic activity of the ribozyme in vitro and for viroid infectivity in vivo, whereas loop 2 has a key opened A at its 3′-side. These structural features promote a specific loop–loop interaction motif across the major groove. The essential features of this tertiary structure element, base pairing between the 5′ U of loop 1 and the 3′ A of loop 2, and interaction of the extrahelical pyrimidine of loop 1 with loop 2, are likely shared by a significant fraction of natural hammerheads.

Transmissible gastroenteritis coronavirus (TGEV) genomic RNA transcription generates 5-and 3-coterminal subgenomic mRNAs. This process involves a discontinuous step during the synthesis of minus-sense RNA that is modulated by transcription-regulating sequences located at the 3 end of the leader (TRS-L) and also preceding each viral gene (TRS-Bs). TRSs include a highly conserved core sequence (CS) (5-CUAAAC-3) and variable flanking sequences. It has been previously proposed that TRS-Bs act as attenuation or stop signals during the synthesis of minus-sense RNAs. The nascent minus-stranded RNA would then be transferred by a template switch process to the TRS-L, which acts as the acceptor RNA. To study whether the TRS-L is structured and to determine whether this structure has a functional impact on genomic and subgenomic viral RNA synthesis, we have used a combination of nuclear magnetic resonance (NMR) spectroscopy and UV thermal denaturation approaches together with site-directed mutagenesis and in vivo transcriptional analyses. The results indicated that a 36-nucleotide oligomer encompassing the wild-type TRS-L forms a structured hairpin closed by an apical AACUAAA heptaloop. This loop contains most of the CS and is isolated from a nearby internal loop by a short Watson-Crick base-paired stem. TRS-L mutations altering the structure and the stability of the TRS-L hairpin affected replication and transcription, indicating the requirement of a functional RNA hairpin structure in these processes.Transmissible gastroenteritis virus (TGEV) is an enveloped virus of the Coronaviridae family containing a single-stranded positive-sense RNA genome of 28.5 kb. The first two-thirds of the genome encodes two replicase polyproteins, while the last third comprises the genes encoding structural and nonstructural viral proteins (Fig. 1A) (10, 18).Coronaviruses (CoVs) share with other plus-stranded RNA viruses the synthesis of subgenomic mRNAs (sgmRNAs) as a mechanism to regulate the expression of the proteins encoded at the 3Ј end of the genome (9,10,18,22,27). CoV sgmRNAs have a terminal leader sequence of around 60 to 93 nucleotides (nt) derived from the 5Ј end of the genome fused to distant RNA coding sequences. This implies a discontinuous step during the synthesis of sgRNAs (22,29,35). This step is guided by transcription-regulating sequences (TRSs) found at the 3Ј end of the leader (TRS-L) and also preceding each gene (TRS-Bs). TRSs comprise a common core sequence (CS) (5Ј-CUAAAC-3Ј), together with 5Ј-and 3Ј-flanking nucleotide sequences that vary depending on the gene but that also are essential for sgmRNA production (29,35).According to the current model of discontinuous transcription in CoVs (26,29,35) (Fig. 1B) and related arteriviruses (23,24,32), the TRS-Bs would act as slow-down and detachment signals for the transcription complex during the synthesis of minus-stranded RNA, possibly by forming a precomplex with the TRS-L and proteins (29). Then, base pairing between the complement of TRS-B (cTRS-B) in the nascent min...

Three-way junction RNAs adopt a recurrent Y shape when two of the helices form a coaxial stack and the third helix establishes one or more tertiary contacts several base pairs away from the junction. In this review, the structure, distribution, and functional relevance of these motifs are examined. Structurally, the folds exhibit conserved junction topologies, and the distal tertiary interactions play a crucial role in determining the final shape of the structures. The junctions and remote tertiary contacts behave as flexible hinge motifs that respond to changes in the other region, providing these folds with switching mechanisms that have been shown to be functionally useful in a variety of contexts. In addition, the juxtaposition of RNA domains at the junction and at the distal tertiary complexes enables the RNA helices to adopt unusual conformations that are frequently used by proteins, RNA molecules, and antibiotics as platforms for specific binding. As a consequence of these properties, Y-shaped junctions are widely distributed in all kingdoms of life, having been observed in small naked RNAs such as riboswitches and ribozymes or embedded in complex ribonucleoprotein systems like ribosomal RNAs, RNase P, or the signal recognition particle. In all cases, the folds were found to play an essential role for the functioning or assembly of the RNA or ribonucleoprotein systems that contain them.