The minus-strand genome of Sendai virus is an assembly of the nucleocapsid protein (N) and RNA, in which each N subunit is associated with precisely 6 nt. Only genomes that are a multiple of 6 nt long replicate efficiently or are found naturally, and their replication promoters contain sequence elements with hexamer repeats. Paramyxoviruses that are governed by this hexamer rule also edit their P gene mRNA during its synthesis, by G insertions, via a controlled form of viral RNA polymerase “stuttering” (pseudo-templated transcription). This stuttering is directed by a cis-acting sequence (3′ UNN UUUUUU CCC), whose hexamer phase is conserved within each virus group. To determine whether the hexamer phase of a given nucleotide sequence within nucleocapsids affected its sensitivity to chemical modification, and whether hexamer phase of the mRNA editing site was important for the editing process, we prepared a matched set of viruses in which a model editing site was displaced 1 nt at a time relative to the genome ends. The relative abilities of these Sendai viruses to edit their mRNAs in cell culture infections were examined, and the ability of DMS to chemically modify the nucleotides of this cis-acting signal within resting viral nucleocapsids was also studied. Cytidines at hexamer phases 1 and 6 were the most accessible to chemical modification, whereas mRNA editing was most extensive when the stutter-site C was in positions 2 to 5. Apparently, the N subunit imprints the nucleotide sequence it is associated with, and affects both the initiation of viral RNA synthesis and mRNA editing. The N-subunit assembly thus appears to superimpose another code upon the genetic code.

We have recently reported an in vitro-evolved precursor tRNA (pre-tRNA) that is able to catalyze aminoacylation on its own 3′-hydroxyl group. This catalytic pre-tRNA is susceptible to RNase P RNA, generating the 5′-leader ribozyme and mature tRNA. The 5′-leader ribozyme is also capable of aminoacylating the tRNA in trans, thus acting as an aminoacyl-tRNA synthetase-like ribozyme (ARS-like ribozyme). Here we report its structural characterization that reveals the essential catalytic core. The ribozyme consists of three stem-loops connected by two junction regions. The chemical probing analyses show that a U-rich region (U59–U62 in J2a/3 and U67–U68 in L3) of the ribozyme is responsible for the recognition of the phenylalanine substrate. Moreover, a GGU-motif (G70–U72) of the ribozyme, adjacent to the U-rich region, forms base pairs with the tRNA 3′ terminus. Our demonstration shows that simple RNA motifs can recognize both the amino acid and tRNA simultaneously, thus aminoacylating the 3′ terminus of tRNA in trans.

The genome of brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, consists of three capped, messenger-sense RNAs. RNA1 and RNA2 encode viral replication proteins 1a and 2a, respectively. RNA3 encodes the 3a movement protein and the coat protein, which are essential for systemic infection in plants but dispensable for RNA3 replication in plants and yeast. A subset of the 250-base intergenic region (IGR), the replication enhancer (RE), contains all cis-acting signals necessary for a crucial, early template selection step, the 1a-dependent recruitment of RNA3 into replication. One of these signals is a motif matching the conserved box B sequence of RNA polymerase III transcripts. Using chemical modification with CMCT, kethoxal, DMS, DEPC, and lead, we probed the structure of the IGR in short, defined transcripts and in full-length RNA3 in vitro, in yeast extracts, and in whole yeast cells. Our results reveal a stable, unbranched secondary structure that is not dependent on the surrounding ORF sequences or on host factors within the cell. Functional 5′ and 3′ deletions that defined the minimal RE in earlier deletion studies map to the end of a common helical segment. The box B motif is presented as a hairpin loop of 7 nt closed by G:C base pairs in perfect analogy to the TΨC-stem loop in tRNAAsp. An adjacent U-rich internal loop, a short helix, and another pyrimidine-rich loop were significantly protected from base modifications. This same arrangement is conserved between BMV and cucumoviruses CMV, TAV, and PSV. In the BMV box B loop sequence, uridines corresponding to tRNA positions T54 and Ψ55 were found to be modified in yeast and plants to 5mU and pseudouridine. Together with the aminoacylated viral 3′-end, this is thus the second RNA replication signal within BMV where the virus has evolved a tRNA structural mimicry to a degree that renders it a substrate for classical tRNA modification reactions in vivo.

By chemical and enzymatic probing, we have analyzed the secondary structure of rodent BC1 RNA, a small brain-specific non-messenger RNA. BC1 RNA is specifically transported into dendrites of neuronal cells, where it is proposed to play a role in regulation of translation near synapses. In this study we demonstrate that the 5′ domain of BC1 RNA, derived from tRNAAla, does not fold into the predicted canonical tRNA cloverleaf structure. We present evidence that by changing bases within the tRNAAla domain during the course of evolution, an extended stem-loop structure has been created in BC1 RNA. The new structural domain might function, in part, as a putative binding site for protein(s) involved in dendritic transport of BC1 RNA within neurons. Furthermore, BC1 RNA contains, in addition to the extended stem-loop structure, an internal poly(A)-rich region that is supposedly single stranded, followed by a second smaller stem-loop structure at the 3′ end of the RNA. The three distinct structural domains reflect evolutionary legacies of BC1 RNA.

The structure of the viral RNA (vRNA) inside intact nucleocapsids of vesicular stomatitis virus was studied by chemical probing experiments. Most of the Watson–Crick positions of the nucleotide bases of vRNA in intact virus and in nucleoprotein (N)-RNA template were accessible to the chemical probes and the phosphates were protected. This suggests that the nucleoprotein binds to the sugar–phosphate backbone of the RNA and leaves the Watson–Crick positions free for the transcription and replication activities of the viral RNA-dependent RNA polymerase. The same architecture has been proposed for the influenza virus nucleocapsids. However, about 5% of the nucleotide bases were found to be relatively nonreactive towards the chemical probes and some bases were hyperreactive. The pattern of reactivities was the same for RNA inside virus and for RNA in N-RNA template that was purified over a CsCl gradient and which had more than 94% of the polymerase and phosphoprotein molecules removed. All reactivities were more or less equal on naked vRNA. This suggests that the variations in reactivity towards the chemical probes are caused by the presence of the nucleoprotein.

We have shown previously that directed hydroxyl radical probing of 16S rRNA from Fe(II) tethered to specific sites within the RNA gives valuable information about RNA–RNA proximities in 70S ribosomes. Here, we extend that study and present probing data from nt 424 in 16S rRNA. To tether an Fe(II) to position 424 in the rRNA we created a specific discontinuity in the RNA by in vitro transcription of the RNA as two separate fragments corresponding to nt 1–423 and 424–1542. An Fe(II)-BABE was covalently attached to a 5′-guanosine-α-phosphorothioate at position 424 and 30S subunits were reconstituted from the two pieces of rRNA and the small subunit proteins. Reconstituted 30S subunits capable of associating with 50S subunits were selected by isolation of 70S ribosomes. Hydroxyl radicals, generated in situ from the tethered Fe(II), cleaved positions in the RNA backbone that were close in three-dimensional space to the Fe(II), and the sites of cleavage were identified using primer extension. Fe(II) tethered to position 424 induces cleavage around nt 424, 513, and 531 in the 5′-domain of 16S rRNA and around nt 1008, 1029, 1044, and 1208 in the 3′-domain of 16S ribosomal RNA. These data constrain the positions of the 420, 1015, 1030 and 1000/1040 helices, for which there is little structural information. Since the 5′- and 3′-domains of 16S rRNA constitute the body and head, respectively, of 30S subunits, these findings provide direct evidence for proximity of RNA elements in the body and head of 30S.

Site-specific photo crosslinking has been used to investigate the RNA neighborhood of 16S rRNA positions U788/U789 in Escherichia coli 30S subunits. For these studies, site-specific psoralen (SSP) which contains a sulfhydryl group on a 17 Å side chain was first added to nucleotides U788/U789 using a complementary guide DNA by annealing and phototransfer. Modified RNA was purified from the DNA and unmodified RNA. For some experiments, the SSP, which normally crosslinks at an 8 Å distance, was derivitized with azidophenacylbromide (APAB) resulting in the photoreactive azido moiety at a maximum of 25 Å from the 4′ position on psoralen (SSP25APA). 16S rRNA containing SSP, SSP25APA or control 16S rRNA were reconstituted and 30S particles were isolated. The reconstituted subunits containing SSP or SSP25APA had normal protein composition, were active in tRNA binding and had the usual pattern of chemical reactivity except for increased kethoxal reactivity at G791 and modest changes in four other regions. Irradiation of the derivatized 30S subunits in activation buffer produced several intramolecular RNA crosslinks that were visualized and separated by gel electrophoresis and characterized by primer extension. Four major crosslink sites made by the SSP reagent were identified at positions U561/U562, U920/U921, C866 and U723; a fifth major crosslink at G693 was identified when the SSP25APA reagent was used. A number of additional crosslinks of lower frequency were seen, particularly with the APA reagent. These data indicate a central location close to the decoding region and central pseudoknot for nucleotides U788/U789 in the activated 30S subunit.

A significant fraction of the bases in a folded, structured RNA molecule participate in noncanonical base pairing interactions, often in the context of internal loops or multi-helix junction loops. The appearance of each new high-resolution RNA structure provides welcome data to guide efforts to understand and predict RNA 3D structure, especially when the RNA in question is a functionally conserved molecule. The recent publication of the crystal structure of the “Loop E” region of bacterial 5S ribosomal RNA is such an event [Correll CC, Freeborn B, Moore PB, Steitz TA, 1997, Cell 91:705–712]. In addition to providing more examples of already established noncanonical base pairs, such as purine–purine sheared pairings, trans-Hoogsteen UA, and GU wobble pairs, the structure provides the first high-resolution views of two new purine–purine pairings and a new GU pairing. The goal of the present analysis is to expand the capabilities of both chemical probing and phylogenetic analysis to predict with greater accuracy the structures of RNA molecules. First, in light of existing chemical probing data, we investigate what lessons could be learned regarding the interpretation of this widely used method of RNA structure probing. Then we analyze the 3D structure with reference to molecular phylogeny data (assuming conservation of function) to discover what alternative base pairings are geometrically compatible with the structure. The comparisons between previous modeling efforts and crystal structures show that the intricate involvements of ions and water molecules in the maintenance of non-Watson–Crick pairs render the process of correctly identifying the interacting sites in such pairs treacherous, except in cases of trans-Hoogsteen A/U or sheared A/G pairs for the adenine N1 site. The phylogenetic analysis identifies A/A, A/C, A/U and C/A, C/C, and C/U pairings isosteric with sheared A/G, as well as A/A and A/C pairings isosteric with both G/U and G/G bifurcated pairings. Thus, each non-Watson–Crick pair could be characterized by a phylogenetic signature of variations between isosteric-like pairings. In addition to the conservative changes, which form a dictionary of pairings isosterically compatible with those observed in the crystal structure, concerted changes involving several base pairs also occur. The latter covariations may indicate transitions between related but distinctive motifs within the loop E of 5S ribosomal RNA.