The well-studied interaction between the MS2 coat protein and its cognate hairpin was used to test the utility of the methylphosphonate linkage as a phosphate analog. A nitrocellulose filter binding assay was used to measure the change in binding affinity upon introduction of a single methylphosphonate stereoisomer at 13 different positions in the RNA hairpin. Comparing these data to the available crystal structure of the complex shows that all phosphates that are in proximity to the protein show a weaker binding affinity when substituted with a phosphorothioate and control positions show no change. However, in two cases, a methylphosphonate isomer either increased or decreased the binding affinity where no interaction can be detected in the crystal structure. It is possible that methylphosphonate substitutions at these positions affect the structure or flexibility of the hairpin. The utility of the methylphosphonate substitution is compared to phosphate ethylation and phosphorothioate substitution experiments previously performed on the same system.

The ribonuclease P ribozyme (RNase P RNA), like other large ribozymes, requires magnesium ions for folding and catalytic function; however, specific sites of metal ion coordination in RNase P RNA are not well defined. To identify and characterize individual nucleotide functional groups in the RNase P ribozyme that participate in catalytic function, we employed self-cleaving ribozyme–substrate conjugates that facilitate measurement of the effects of individual functional group modifications. The self-cleavage rates and pH dependence of two different ribozyme–substrate conjugates were determined and found to be similar to the single turnover kinetics of the native ribozyme. Using site-specific phosphorothioate substitutions, we provide evidence for metal ion coordination at the pro-RP phosphate oxygen of A67, in the highly conserved helix P4, that was previously suggested by modification-interference experiments. In addition, we detect a new metal ion coordination site at the pro-SP phosphate oxygen of A67. These findings, in combination with the proximity of A67 to the pre-tRNA cleavage site, support the conclusion that an important role of helix P4 in the RNase P ribozyme is to position divalent metal ions that are required for catalysis.

Eukaryotic translation initiation factor 4A (eIF4A) has been proposed to use the energy of ATP hydrolysis to remove RNA structure in the 5′ untranslated region (UTR) of mRNAs, helping the 43S ribosomal complex bind to an mRNA and scan to find the 5′-most AUG initiator codon. We have examined the effect of changing the atomic composition and length of single-stranded oligonucleotides on binding to eIF4A and on stimulation of its ATPase activity once bound. Substitution of 2′-OH groups with 2′-H or 2′-OCH3 groups reduces ATPase stimulation at least 100-fold, to background levels, without significantly affecting oligonucleotide affinity. These effects suggest that 2′-OH groups participate in an eIF4A conformational change that occurs subsequent to oligonucleotide binding and is required for ATPase stimulation. Replacing nonbridging oxygen atoms in phosphodiester linkages with sulfur atoms to make phosphorothioate linkages has no significant effect on stimulation, while substantially increasing affinity. Extending the length of an RNA oligonucleotide from 4 to ∼15 nt gradually increases oligonucleotide affinity and ATPase stimulation. Consistent with this observation, the increase in affinity and stimulation provided by phosphorothioate linkages and 2′-OH groups is proportional to the number of these groups present within larger oligonucleotides. Further, changing the position of blocks of phosphorothioate linkages or 2′-OH groups within a larger oligonucleotide does not affect affinity and has only a small effect on stimulation. These observations suggest that numerous interactions between the oligonucleotide and eIF4A contribute individually to binding and ATPase stimulation. Nevertheless, significant stimulation is observed with as few as four RNA residues. These properties may allow eIF4A to operate within regions of 5′ UTRs containing only short stretches of exposed single-stranded RNA. As stimulation increases when longer stretches of single-stranded RNA are available, it is possible that the accessibility of single-stranded RNA in a 5′ UTR influences translation efficiency.

The use of T7 RNA polymerase to prepare large quantities of RNA of a particular sequence has greatly facilitated the study of both the structure and function of RNA. Generally, it has been believed that the products of this technique are highly homogeneous in sequence, with only a few noted exceptions. We have carefully examined the transcriptional products of several tRNAs that vary in their 5′ end sequence and found that, for those molecules that begin with multiple, consecutive guanosines, the transcriptional products are far from homogenous. Although a template beginning with GCG showed no detectable 5′ end heterogeneity, two tRNA templates designed to have either four or five consecutive guanosines at their 5′ ends had more than 30% of their total transcriptional products extended by at least one untemplated nucleotide at their 5′ end. By simply reducing the number of consecutive guanosines, the heterogeneity was reduced significantly. The presence of this 5′ end heterogeneity in combination with the 3′ end heterogeneity common to T7 transcriptions results in a mixture of RNA molecules even after rigorous size purification.

The RNA subunit of bacterial ribonuclease P is a catalytic RNA that cleaves precursor tRNAs to generate mature tRNA 5′ ends. A self-cleaving RNase P RNA–substrate conjugate was used in modification-interference analysis to identify purine N-7 and ribose 2′-hydroxyl functional groups that are critical to catalysis. We identify six adenine N-7 groups and only one 2′-hydroxyl that, when substituted with 7-deazaadenine or 2′-deoxy analogues, respectively, reduce the RNase P catalytic rate approximately 10-fold at pH 8 and limiting concentration of magnesium. Two sites of low-level interference by phosphorothioate modification were detected in addition to the four sites of strong interference documented previously. These modification-interference results, the absolute phylogenetic conservation of these functional groups in bacterial RNase P RNA, their proximity to the substrate–phosphate in the tertiary structure of the ribozyme–substrate complex, and the importance of some of the sites for binding of catalytic magnesium all implicate these functional groups as components of the RNase P active site. Five of the 7-deazaadenine interferences are suppressed at pH 6, where the hydrolytic step is rate-limiting, or at saturating concentrations of magnesium. We propose, therefore, that these base functional groups are specifically engaged in the catalytic center of RNase P RNA, possibly by involvement in magnesium-dependent folding. One 7-deazaadenine interference and one 2′-deoxy-interference, although partially suppressed at pH 6, are not suppressed at saturating magnesium concentrations. This implicates these groups in magnesium-independent folding of the catalytic substructure of the ribozyme.

The two transesterification reactions catalyzed by self-splicing group II introns take place in either two active sites or two conformations of a single active site involving rearrangements of the positions of the reacting groups. We have investigated the effects on the rates of the chemical steps of the two reactions due to sulfur substitution of nonbridging oxygens at both the 5′ and 3′ splice sites as well as the deoxyribose substitution of the ribose 2′ hydroxyl group at the 5′ splice site. The data suggest that the two active sites differ in their interactions with several of these groups. Specifically, sulfur substitution of the pro-Sp nonbridging oxygen at the 5′ splice site reduces the chemical rate of the step one branching reaction by at least 250-fold, whereas substitution of the pro-Sp oxygen at the 3′ splice site has only a 4.5-fold effect on the chemical rate of step two. Previous work demonstrated that the Rp phosphorothioate substitutions at both the 5′ and 3′ splice sites reduced the rate of both steps of splicing to an undetectable level. These results suggest that either two distinct active sites catalyze the two steps or that more significant alterations must be made in a single bifunctional active site to accommodate the two different reactions.