Insertion of selenocysteine into a growing peptide requires the unusual tRNASec (Zinoni et al., 1987; Stadtman, 1990; Böck et al., 1991). This tRNA has an extended D-stem containing six base pairs, which, in the case of eukaryotic tRNASec (euk-tRNASec), is the key identity element for selenylation and phosphorylation (Wu & Gross, 1994; Amberg et al., 1996). Two secondary structures have been proposed for the euk-tRNASec, which differ in the base pairing of the acceptor/T helical domain (Diamond et al., 1981; Böck et al., 1991; Sturchler et al., 1993). One structure has the normal seven base pairs in the acceptor stem and five base pairs in the T-stem (7/5 structure, Fig. 1, left), and is characterized by an unusually long four-nucleotide unpaired region between the acceptor and D-stems (Connector 1) and an unpaired nucleotide, C64a, in the T-stem. The alternate structure features the normal two nucleotides in Connector 1 and a 13-base pair acceptor/T domain comprised of nine base pairs in the acceptor stem and four in the T-stem (9/4 structure, Fig. 1, right). This 9/4 structure was initially proposed by analogy with the prokaryotic tRNASec (prok-tRNASec), which also contains 13 base pairs in the acceptor/T helical domain. However, in this case, there are eight and five base pairs in the acceptor and T-stems, respectively. The acceptor/T helical domain having 13 base pairs is thought to be a key structural element determining the functionalities pattern of tRNASec in both prokaryotes and eukaryotes (Böck et al., 1991).

Histone pre-mRNA 3′ processing is controlled by a hairpin element preceding the processing site that interacts with a hairpin-binding protein (HBP) and a downstream spacer element that serves as anchoring site for the U7 snRNP. In addition, the nucleotides following the hairpin and surrounding the processing site (ACCCA′CA) are conserved among vertebrate histone genes. Single to triple nucleotide mutations of this sequence were tested for their ability to be processed in nuclear extract from animal cells. Changing the first four nucleotides had no qualitative and little if any quantitative effects on histone RNA 3′ processing in mouse K21 cell extract, where processing of this gene is virtually independent of the HBP. A gel mobility shift assay revealing HBP interactions and a processing assay in HeLa cell extract (where the contribution of HBP to efficient processing is more important) showed that only one of these mutations, predicted to extend the hairpin by one base pair, affected the interaction with HBP. Mutations in the next three nucleotides affected both the cleavage efficiency and the choice of processing sites. Analysis of these novel sites indicated a preference for the nucleotide 5′ of the cleavage site in the order A > C > U > G. Moreover, a guanosine in the 3′ position inhibited cleavage. The preference for an A is shared with the cleavage/polyadenylation reaction, but the preference order for the other nucleotides is different [Chen F, MacDonald CC, Wilusz J, 1995, Nucleic Acids Res 23:2614–2620].

Experimental results are presented suggesting that 23S rRNA is directly involved in the peptide bond formation usually performed on the ribosome. Although several reports have indicated that the eubacterial peptidyltransferase reaction does not necessarily require all the ribosomal proteins, the reconstitution of peptidyltransferase activity by a naked 23S rRNA without the help of any of the ribosomal proteins has not been reported previously. It is demonstrated that an E. coli 23S rRNA transcript synthesized by T7 RNA polymerase in vitro was able to promote peptide bond formation in the presence of 0.5% SDS. The reaction was inhibited by the peptidyltransferase-specific antibiotics chloramphenicol and carbomycin, and by digestion with RNases A and T1. Site-directed mutageneses at two highly conserved regions close to the peptidyltransferase center ring, G2252 to U2252 and C2507G2581 to U2507A2581, also suppressed peptide bond formation. These findings strongly suggest that 23S rRNA is the peptidyltransferase itself.

In the course of evolving variants of the Tetrahymena thermophila Group I ribozyme for improved DNA cleavage in vitro, we witnessed the unexpected emergence of a derived molecular species, capable of acting as a partner for the ribozyme, but no longer autocatalytic. This new RNA species exhibits a deletion in the catalytic core and participates in a productive intermolecular interaction with an active ribozyme, thus insuring its survival in the population. These novel RNA molecules have evolved a precise catalytic interaction with the Group I ribozyme and depend for their survival on the continued presence of active catalysts. This interaction hints at the complexity that may inevitably arise even in simple evolving systems.

The CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase] catalyzes the addition and regeneration of the 3′-terminal CCA sequence of tRNAs. We show that the CCA-adding enzyme will specifically add a CCA terminus to synthetic full-length tDNA and to DNA oligonucleotides corresponding to the “top half” of tRNA — the acceptor stem and TψC stem-loop of tRNA. CCA addition to the top half tDNA minihelices requires a 2′ as well as a 3′ OH at the 3′ terminus of the tDNA. Addition also depends on the length of the base paired stem, and is facilitated by, but is not dependent upon, the presence of a TψC loop. These results provide further evidence for independent functions of the top and bottom halves of tRNA, and support the hypothesis that these two structurally distinct and functionally independent domains evolved independently.

The U3 snoRNA coding sequences from the genomic DNAs of Kluyveromyces delphensis and four variants of the Kluyveromyces marxianus species were cloned by PCR amplification. Nucleotide sequence analysis of the amplification products revealed a unique U3 snoRNA gene sequence in all the strains studied, except for K. marxianus var. fragilis. The K. marxianus U3 genes were intronless, whereas an intron similar to those of the Saccharomyces cerevisiae U3 genes was found in K. delphensis. Hence, U3 genes with and without intron are found in yeasts of the Saccharomycetoideae subfamily. The secondary structure of the K. delphensis pre-U3 snoRNA and of the K. marxianus mature snoRNAs were studied experimentally. They revealed a strong conservation in yeasts of (1) the architecture of U3 snoRNA introns, (2) the 5′-terminal domain of the mature snoRNA, and (3) the protein-anchoring regions of the U3 snoRNA 3′ domain. In contrast, stem-loop structures 2, 3, and 4 of the 3′ domain showed great variations in size, sequence, and structure. Using a genetic test, we show that, in spite of these variations, the Kluyveromyces U3 snoRNAs are functional in S. cerevisiae. We also show that S. cerevisiae U3A snoRNAs lacking the stem-loop structure 2 or 4 are functional. Hence, U3 snoRNA function can accommodate great variations of the RNA 3′-terminal domain.

Efficient transcription termination downstream of poly(A) sites has been shown to correlate with the strength of an upstream polyadenylation signal and the presence of a polymerase pause site. To further investigate the mechanism linking termination with 3′-end processing, we analyzed the cis-acting elements that contribute to these events in the Saccharomyces cerevisiae FBP1 gene. FBP1 has a complex polyadenylation signal, and at least three efficiency elements must be present for efficient processing. However, not all combinations of these elements are equally effective. This gene also shows a novel organization of sequence elements. A strong positioning element is located upstream, rather than downstream, of the efficiency elements, and functions to select the cleavage site in vitro and in vivo. Transcription run-on analysis indicated that termination occurs within 61 nt past the poly(A) site. Deletion of two UAUAUA-type efficiency elements greatly reduces polyadenylation in vivo and in vitro, but transcription termination is still efficient, implying that FBP1 termination signals may be distinct from those for polyadenylation. Alternatively, assembly of a partial, but nonfunctional, polyadenylation complex on the nascent transcript may be sufficient to cause termination.

The 5′ untranslated region (UTR) of the long-lived Escherichia coli ompA transcript functions as an mRNA stabilizer that can prolong the cytoplasmic lifetimes of a variety of messages to which it is fused. Previous studies have identified two domains of this 5′ UTR that together are responsible for its stabilizing effect. One is a 5′-terminal stem-loop. The other is a single-stranded RNA segment (ss2) that contains a ribosome binding site highly complementary to 16S ribosomal RNA. Here we report a detailed investigation of the function of these two stabilizing elements. Our data indicate that mRNA protection by a 5′ stem-loop requires no sequence features or thermodynamic stability beyond the minimum necessary for stem-loop formation. Stabilization by ss2 appears to result not from a high frequency of translation initiation, but rather from a high degree of occupancy of this 5′ UTR segment by bound ribosomes. Although close spacing of translating ribosomes is not critical for message stabilization by the ompA 5′ UTR, mRNA longevity does require the periodic passage of ribosomes through the protein-coding region. Unlike bound ribosomes, which hinder mRNA cleavage by RNase E, the 5′ stem-loop appears to impede degradation of ompA mRNA via a distinct pathway that is RNase E-independent. These findings imply that the ompA 5′ UTR prolongs mRNA longevity by impeding multiple pathways for mRNA degradation.

The N-terminal RNA binding domain of the human U1A protein (RBD1) specifically binds an RNA hairpin of U1 snRNA as well as two internal loops in the 3′ UTR of its own mRNA. Here, a single cysteine has been introduced into Loop 1 of RBD1, which is subsequently used to attach (EDTA-2-aminoethyl) 2-pyridyl disulfide-Fe3+ (EPD-Fe). This EDTA-Fe derivative is used to generate hydroxyl radicals to cleave the proximal RNA sugar–phosphate backbone in the RNA–RBD complexes. RBD1(K20C)–EPD-Fe cleaves the 5′ strand of the RNA hairpin stem, centered four base pairs away from the base of the loop, and cleaves the UTR in two places, again centered on the 5′ side of the fourth base pair from each internal loop. These data, extrapolated to the position of Lys 20 in RBD1, orient the two proteins bound to the UTR, and provide direct biochemical evidence for the proposed model of the RBD1:UTR complex.

We have synthesized the analogue 2′-deoxy-2′-thio-CTP (CTP-SH) and tested its ability to support RNA transcription in place of CTP. The modified nucleotide in a transcription reaction and in the absence of CTP generated the appropriately sized fragment when a mutant T7 polymerase (Y639F) was used. Wild-type polymerase was unable to generate RNA under the same conditions. Transcription was optimal around pH 7.5 and was dependent upon CTP-SH concentration. Transcripts containing the analogue were efficiently isolated using a thiol-activated sepharose column. Insertion of CTP-SH into the HDV ribozyme, replacing all cytidine residues with 2′-thiocytidine, appears to inhibit self-cleaving activity, even in the presence of manganese. The ability to introduce the CTP-SH analogue enzymatically into RNA opens the way for new structure–function studies where the 2′-hydroxyl can be efficiently replaced by a thiol group.