Interaction between the antisense and target RNAs involved in the regulation of IncB plasmid replication.

Physical analysis of RNA I, the small antisense RNA which regulates the replication of IncB miniplasmid pMU720, showed that it is a highly structured molecule containing an imperfectly paired stem closed by a 6-base hairpin loop. Mutational studies revealed that a 3-base sequence in the hairpin loop is critical to the interaction between RNA I and its complementary target in the RepA mRNA (RNA II). Furthermore, a 2-base interior loop in the upper stem was found to play an important role in facilitating effective binding between RNA I and RNA II. From these analyses, a model describing the molecular mechanism of binding between RNA I and RNA II is proposed.     

Dramatic size variation of yeast mitochondrial RNAs suggests that RNase P RNAs can be quite small

The gene coding for the AU-rich RNA required for mitochondrial RNase P activity in Saccharomyces cerevisiae codes for a 490-base RNA while that in Candida glabrata codes for a 227-base RNA. We have detected a 140-nucleotide RNA coded by the mitochondrial DNA from Saccharomycopsis fibuligera by hybridization with an oligonucleotide complementary to a conserved sequence found in mitochondrial and prokaryotic RNase P RNAs. DNA sequence analysis of the mitochondrial DNA from the region coding for this RNA revealed a second conserved sequence block characteristic of RNase P RNA genes and the presence of a downstream tRNA(Pro) gene. Like previously characterized mitochondrial RNase P RNAs, this small RNA is extremely AU-rich. The discovery of this 140-base RNA suggests that naturally occurring RNase P RNAs may be quite small.     

RNA catalytic properties of the minimum (-)sTRSV sequence

We have identified an RNA catalytic domain within the sequence of the 359 base long negative-strand satellite RNA of tobacco ringspot virus. The catalytic domain contains two minimal sequences of satellite RNA, a 50-base catalytic RNA sequence, and a 14-base substrate RNA sequence. The catalytic complex of catalytic RNA/substrate RNA represents a structure not previously found in any RNA catalytic reaction described to date. The reaction is truly catalytic since the catalytic RNA has multiple substrate cleavage events and is not consumed during the course of the reaction. A linear relationship is seen between reaction rate and catalytic RNA concentration. The reaction has a Km of 0.03 microM, a kcat of 2.1/min, a temperature optimum of near 37 degrees C, and an energy of activation of 19 kcal/mol.     

Minimal Rev-response element for type 1 human immunodeficiency virus.

Rev protein regulates nuclear export of viral mRNAs that contain a 240-base RNA sequence termed the Rev-response element (RRE). We demonstrate that an 88-base truncated RRE encompassing a known Rev binding site can mediate Rev responsiveness in vivo. Two tandem copies of this mutant function as efficiently as the full-length RRE.     

Molecular cloning of the Harvey sarcoma virus circular DNA intermediates. II. Further structural analyses.

Three species of unintegrated supercoiled Harvey sarcoma virus DNA (6.6, 6.0, and 5.4 kilobase pairs) have been molecularly cloned from Harvey sarcoma virus-infected cells. On the basis of restriction enzyme analyses, the 6.6- and 6.0-kilobase pair viral DNAs contain two and one copies, respectively, of a 650-base pair DNA segment which contains sequences present at the 3' and 5' termini of the viral genome. R-loop structures formed between Moloney leukemia virus RNA and the cloned Harvey sarcoma virus DNA indicated that about 500 base pairs of the 650-base pair repeating segment was complementary to the 3' end of the viral RNA. During amplification in the Escherichia coli host, some recombinants containing the 6.6- or the 6.0-kilobase pair Harvey sarcoma virus DNA insert acquired or lost the complete 650-base pair DNA segment. These changes occurred in both recA+ and recA- E. coli.     

Secondary structure determination of the conserved 98-base sequence at the 3' terminus of hepatitis C virus genome RNA.

The RNA genome of hepatitis C virus (HCV) terminates with a highly conserved 98-base sequence. Enzymatic and chemical approaches were used to define the secondary structure of this 3'-terminal element in RNA transcribed in vitro from cloned cDNA. Both approaches yielded data consistent with a stable stem-loop structure within the 3'-terminal 46 bases. In contrast, the 5' 52 nucleotides of this 98-base element appear to be less ordered and may exist in multiple conformations. Under the experimental conditions tested, interaction between the 3' 98 bases and upstream HCV sequences was not detected. These data provide valuable information for future experiments aimed at identifying host and/or viral proteins which interact with this highly conserved RNA element.     

Syntheses of alternating oligo-2'-O-methylribonucleoside methylphosphonates and their interactions with HIV TAR RNA

Oligonucleotide analogues 15-20 nucleotides in length have been prepared, whose sequences are complementary to nucleotides in the upper hairpin of HIV TAR RNA. These alternating oligonucleoside methylphosphonates, mr-AOMPs, contain 2'-O-methylribonucleosides and alternating methylphosphonate and phosphodiester internucleotide linkages. The methylphosphonate and phosphodiester linkages of these oligomers are highly resistant to hydrolysis by exonuclease activity found in mammalian serum and to endonucleases, such as S1 nuclease. The oligomers were prepared using automated phosphoramidite chemistry and terminate with a 5'-phosphate group, which provides an affinity handle for purification by strong anion exchange HPLC. A 15-mer mr-AOMP, 1676, that is complementary to the 5'-side of the TAR RNA hairpin, including the 3-base bulge and 6-base loop region, forms a 1:1 duplex with a complementary RNA 18-mer, mini-TAR RNA. The T(m) of this duplex is 71 degrees C, which is similar to that of the duplex formed by the corresponding all phosphodiester 15-mer. Introduction of two mismatched bases reduces the T(m) by 17 degrees C. The apparent dissociation constant, K(d), for the 1676/mini-TAR RNA duplex as determined by an electrophoretic mobility shift assay at 37 degrees C is 0.3 nM. Oligomer 1676 also binds tightly to the full length TAR RNA target under physiological conditions (K(d) = 20 nM), whereas no binding was observed by the mismatched oligomer. A 19-mer that is complementary to the entire upper hairpin also binds to TAR RNA with a K(d) that is similar to that of 1676, a result that suggests only part of the oligomer binds. When two of the methylphosphonate linkages in the region complementary to the 6-base loop are replaced with phosphodiester linkages, the K(d) is reduced by approximately a factor of 10. This result suggests that interactions between TAR RNA and the oligomer occur initially with nucleotides in the 6-base loop, and that these interactions are sensitive to presence and possibly the chirality of the methylphosphonate linkages in the oligomer. The high affinities of mr-AOMPs for TAR RNA and their resistance to nuclease hydrolysis suggests their potential utility as antisense agents in cell culture.     

In vitro analysis of the interaction between the FinO protein and FinP antisense RNA of F-like conjugative plasmids

The FinO protein regulates the transfer potential of F-like conjugative plasmids through its interaction with FinP antisense RNA and its target, traJ mRNA. FinO binds to and protects FinP from degradation and promotes duplex formation between FinP and traJ mRNA in vitro. The FinP secondary structure consists of two stem-loop domains separated by a 4-base spacer and terminated by a 6-base tail. Previous studies suggested FinO bound to the smooth 14-base pair helix of stem-loop II. In this investigation, RNA mobility shift analysis was used to study the interaction between a glutathione S-transferase (GST)-FinO fusion protein and a series of synthetic FinP and traJ mRNA variants. Mutations in 16 of the 28 bases in stem II of FinP that are predicted to disrupt base pairing did not significantly alter the GST-FinO binding affinity. Removal of the single-stranded regions on either side of stem-loop II led to a dramatic decrease in GST-FinO binding to FinP and to the complementary region of the traJ mRNA leader. While no evidence for sequence-specific contacts was found, the results suggest that FinO recognizes the overall shape of the RNA and is influenced by the length of the single-stranded regions flanking the stem-loop.     

RNA replication from the simian virus 5 antigenomic promoter requires three sequence-dependent elements separated by sequence-independent spacer regions

We have previously shown for the paramyxovirus simian virus 5 (SV5) that a functional promoter for RNA replication requires proper spacing between two discontinuous elements: a 19-base segment at the 3' terminus (conserved region I [CRI]) and an 18-base internal region (CRII) that is contained within the coding region of the L protein gene. In the work described here, we have used a reverse-genetics system to determine if the 53-base segment between CRI and CRII contains additional sequence-specific signals required for optimal replication or if this segment functions solely as a sequence-independent spacer region. A series of copyback defective interfering minigenome analogs were constructed to contain substitutions of nonviral sequences in place of bases 21 to 72 of the antigenomic promoter, and the relative level of RNA replication was measured by Northern blot analysis. The results from our mutational analysis indicate that in addition to CRI and CRII, optimal replication from the SV5 antigenomic promoter requires a third sequence-dependent element located 51 to 66 bases from the 3' end of the RNA. Minigenome RNA replication was not affected by changes in the either the position of this element in relation to CRI and CRII or the predicted hexamer phase of NP encapsidation. Thus, optimal RNA replication from the SV5 antigenomic promoter requires three sequence-dependent elements, CRI, CRII and bases 51 to 66.     

Ricin A-chain inhibitors resembling the oxacarbenium ion transition state

Ricin toxin A-chain (RTA) is expressed by the castor bean plant and is among the most potent mammalian toxins. Upon activation in the cytosol, RTA depurinates a single adenine from position 4324 of rat 28S ribosomal RNA, causing inactivation of ribosomes by preventing the binding of elongation factors. Kinetic isotope effect studies have established that RTA operates via a D(N)*A(N) mechanism involving an oxacarbenium ion intermediate with bound adenine [Chen, X.-Y., Berti, P. J., and Schramm, V. L. (2000) J. Am. Chem. Soc. 122, 1609-1617]. On the basis of this information, stem-loop RNA molecules were chemically synthesized, incorporating structural features of the oxacarbenium ion-like transition state. A 10-base RNA stem-loop incorporating (1S)-1-(9-deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol at the depurination site binds four times better (0.57 microM) than the 10-base RNA stem-loop with adenosine at the depurination site (2.2 microM). A 10-base RNA stem-loop with 1,2-dideoxyribitol [(2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran] at the depurination site binds with a Kd of 3.2 microM and tightens to 0.75 microM in the presence of 9-deazaadenine. A similar RNA stem-loop with 1,4-dideoxy-1,4-imino-D-ribitol at the depurination site binds with a K(d) of 1.3 microM and improves to 0.65 micro;M with 9-deazaadenine added. When (3S,4R)-4-hydroxy-3-(hydroxymethyl)pyrrolidine was incorporated at the depurination site of a 14-base RNA stem-loop, the Kd was 0.48 microM. Addition of 9-deazaadenine tightens the binding to 0.10 microM whereas added adenine increases the affinity to 12 nM. The results of this study are consistent with the unusual dissociative D(N)*A(N) mechanism determined for RTA. Knowledge of this intermediate has led to the design and synthesis of the highest affinity inhibitor reported for the catalytic site of RTA.