Exploration of the L18 binding site on 5S RNA by deletion mutagenesis.

Several deletion variants of E. coli 5S RNA have been constructed and produced either in vivo or in vitro using T7 RNA Polymerase. Their structures and ribosomal protein L18 binding properties have been examined. All of them are similar to wild-type 5S RNA in their helix II-III regions, where L18 binds [Huber, P.W. and Wool, I.G. (1984) Proc. Natl. Acad. Sci. (USA) 81, 322-326; Douthwaite, S., Christensen, A., and Garrett, R.A. (1982) Biochemistry 21, 2313-2320.], by NMR criteria. However, none of the molecules examined that lack the helix IV-helix V stem bind L18 efficiently, even though that portion of 5S RNA is outside the L18 footprint. The L18 binding site is clearly more than a simple hairpin loop.     

Imino proton exchange in the 5S RNA of Escherichia coli and its complex with protein L25 at 490 MHz

Imino proton exchange has been examined by NMR in the 5S RNA of Escherichia coli, its principal RNase A resistant fragment, fragment 1 (bases 1-11, 69-120), and complexes between that fragment and ribosomal protein L25 by using both real-time and relaxation techniques. Fragment 1 RNA imino protons exchange at rates between 0.5 and 15 s-1 at 303 K in 5 mM cacodylate buffer, pH 7.4. In contrast with many tRNAs, intact 5S RNA contains no imino protons with exchange lifetimes as great as 1 min. Consistent with the results of Gueron and his colleagues [Leroy, J. L., Bolo, N., Figueroa, N., Plateau, P., & Gueron, M. (1985) J. Biomol. Struct. Dyn. 2,915-939; Leroy, J. L., Broseta, D., & Gueron, M. (1985) J. Mol. Biol. 184, 165-178] with tRNA, exchange in 5S RNA is catalyst-limited under conditions generally used for imino proton spectroscopy, such as those given above. Using Gueron's catalyst saturation technique, base pair opening rates have been measured for several AU and GU base pairs in fragment 1. They range from 50 to 300 s-1 at 303 K and depend on base pair type and also to some degree on context. Similar studies have been done on complexes of L25 and fragment 1. The binding of L25 to fragment 1 reduces the exchange rate of many imino protons within the region to which it binds, consistent with the hypothesis that its binding stabilizes the secondary structure of 5S RNA.     

Minor groove orientation for the (1S,2R,3S,4R)-N2- [1-(1,2,3,4-tetrahydro-2,3,4-trihydroxybenz[a]anthracenyl)]-2'-deoxyguanosyl adduct in the N-ras codon 12 sequence

The conformation of the trans-anti-(1S,2R,3S,4R)-N(2)-[1-(1,2,3,4-tetrahydro-2,3,4-trihydroxybenz[a]anthracenyl)]-2'-deoxyguanosyl adduct in d(G(1)G(2)C(3)A(4)G(5)X(6)T(7)G(8)G(9)T(10)G(11)).d(C(12)A(13)C(14)C(15)A(16)C(17)C(18)T(19)G(20)C(21)C(22)), bearing codon 12 of the human N-ras protooncogene (underlined), was determined. This adduct had S stereochemistry at the benzylic carbon. Its occurrence in DNA is a consequence of trans opening by the deoxyguanosine amino group of (1R,2S,3S,4R)-1,2-epoxy-1,2,3,4-tetrahydrobenz[a]anthracenyl-3,4-diol. The resonance frequencies, relative to the unmodified DNA, of the X(6) H1' and H6 protons were shifted downfield, whereas those of the C(18) and T(19) H1', H2', H2' ', and H3' deoxyribose protons were shifted upfield. The imino and amino resonances exhibited the expected sequential connectivities, suggesting no interruption of Watson-Crick pairing. A total of 426 interproton distances, including nine uniquely assigned BA-DNA distances, were used in the restrained molecular dynamics calculations. The refined structure showed that the benz[a]anthracene moiety bound in the minor groove, in the 5'-direction from the modified site. This was similar to the (+)-trans-anti-benzo[a]pyrene-N(2)-dG adduct having S stereochemistry at the benzylic carbon [Cosman, M., De Los Santos, C., Fiala, R., Hingerty, B. E., Singh, S. B., Ibanez, V., Margulis, L. A., Live, D., Geacintov, N. E., Broyde, S., and Patel, D. J. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1914-1918]. It differed from the (-)-trans-anti-benzo[c]phenanthrene-N(2)-dG adduct having S stereochemistry at the benzylic carbon, which intercalated in the 5'-direction [Lin, C. H., Huang, X., Kolbanovskii, A., Hingerty, B. E., Amin, S., Broyde, S., Geacintov, N. E., and Patel, D. J. (2001) J. Mol. Biol. 306, 1059-1080]. The results provided insight into how PAH molecular topology modulates adduct structure in duplex DNA.     

Nuclear Overhauser experiments at 500 MHz on the downfield proton spectrum of a ribonuclease-resistant fragment of 5S ribonucleic acid

The downfield (9-15 ppm) proton NMR spectrum of a RNase A resistant fragment of E. coli 5S RNA has been studied by nuclear Overhauser methods. The fragment comprises bases 1-11 and 69-120 of the parent molecule [Douthwaite, S., Garrett, R.A., Wagner, R., & Feunteun, J. (1979) Nucleic Acids Res. 6, 2453-2470]. The nuclear Overhauser data identify two double helical structures in the fragment whose sequences are (GC)3(AU)(GC)3 and (GC)2(AU)(GU). These structures correspond exactly to the central portions of the terminal stem and procaryotic loop helices which should exist in the fragment sequences according to the Fox-Woese model [Fox, G.E., & Woese, C. R. (1975) Nature (London) 256, 505-506] of 5S RNA secondary structure. The significance of these and other nuclear Overhauser effects detected for the structure of 5S RNA and its fragment is discussed.     

Functional analyses of positions across the 5' splice site of the trypanosomatid spliced leader RNA. Implications for base-pair interaction with U5 and U6 snRNAs

In this study, we have used a genetic compensatory approach to examine the functional significance of the previously proposed interaction of spliced leader (SL) RNA with U5 small nuclear RNA (snRNA) (Dungan, J. D., Watkins, K. P., and Agabian, N. (1996) EMBO J. 15, 4016-4029; Xu, Y.-X., Ben Shlomo, H., and Michaeli, S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8473-8478) and the interaction of the SL RNA intron with U6 snRNA analogous to cis-splicing. Mutations were introduced at positions -4, -1, +1, +4, +5, and +7/+8 relative to the SL RNA 5' splice site that were proposed to interact with U5 and U6 snRNAs. All mutants exhibited altered splicing phenotypes compared with the parental strain, showing the importance of these intron and exon positions for trans-splicing. Surprisingly, mutation at invariant +1 position did not abolish splicing completely, unlike cis-splicing, but position +2 had the most severe effect on trans-splicing. Compensatory mutations were introduced in U5 and U6 snRNAs to examine whether the defects resulted from failure to interact with these snRNAs by base pairing. Suppression was observed only for positions +5 and +7/+8 with U5 compensatory mutations and for position +5 with a U6 compensatory mutation, supporting the existence of a base pair interaction of U5 and U6 with the SL RNA intron region. The failure to suppress the other SL RNA mutants by the U5 compensatory mutations suggests that another factor(s) interacts with these key SL RNA positions.     

Structure of an oligodeoxynucleotide containing a butadiene oxide-derived N1 beta-hydroxyalkyl deoxyinosine adduct in the human N-ras codon 61 sequence

The solution structure of the N1-(1-hydroxy-3-buten-2(S)-yl)-2'-deoxyinosine adduct arising from the alkylation of adenine N1 by butadiene epoxide (BDO), followed by deamination to deoxyinosine, was determined, in the oligodeoxynucleotide d(CGGACXAGAAG).d(CTTCTCGTCCG). This oligodeoxynucleotide contained the BDO adduct at the second position of codon 61 of the human N-ras protooncogene, and was named the ras61 S-N1-BDO-(61,2) adduct. (1)H NMR revealed a weak C(5) H1' to X(6) H8 NOE, followed by an intense X(6) H8 to X(6) H1' NOE. Simultaneously, the X(6) H8 to X(6) H3' NOE was weak. The resonance arising from the T(17) imino proton was not observed. (1)H NOEs between the butadiene moiety and the DNA positioned the adduct in the major groove. Structural refinement based upon a total of 364 NOE-derived distance restraints yielded a structure in which the modified deoxyinosine was in the high syn conformation about the glycosyl bond, and T(17), the complementary nucleotide, was stacked into the helix, but not hydrogen bonded with the adducted inosine. The refined structure provided a plausible hypothesis as to why this N1 deoxyinosine adduct strongly coded for the incorporation of dCTP during trans lesion DNA replication, both in Escherichia coli [Rodriguez, D. A., Kowalczyk, A., Ward, J. B. J., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2001) Environ. Mol. Mutagen. 38, 292-296], and in mammalian cells [Kanuri, M., Nechev, L. N., Tamura, P. J., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2002) Chem. Res. Toxicol. 15, 1572-1580]. Rotation of the N1 deoxyinosine adduct into the high syn conformation may facilitate incorporation of dCTP via Hoogsteen-type templating with deoxyinosine, thus generating A-to-G mutations.     

Intercalation of the (1R,2S,3R,4S)-N6-[1-(1,2,3,4-tetrahydro-2,3,4-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl adduct in the N-ras codon 61 sequence: DNA sequence effects

The structure of the bay region (1R,2S,3R,4S)-N6-[1-(1,2,3,4-tetrahydro-2,3,4-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl adduct at X(7) of 5'-d(CGGACAXGAAG)-3'.5'-d(CTTCTTGTCCG)-3', incorporating codons 60, 61 (underlined), and 62 of the human N-ras protooncogene, was determined by NMR. This was the bay region benz[a]anthracene RSRS (61,3) adduct. The BA moiety intercalated above the 5'-face of the modified base pair. NOE connectivities between imino protons were disrupted at T16 and T17. Large chemical shifts at the lesion site were consistent with ring current shielding arising from the BA moiety. A large chemical shift dispersion was observed for the BA aromatic protons. An increased rise of 8.17 A was observed between base pairs A6 x T17 and X7 x T(16). The PAH moiety stacked with the purine ring of A6, the 5'-neighbor nucleotide. This resulted in buckling of the 5'-neighbor A6 x T17 base pair, evidenced by exchange broadening for the T17 imino resonance. It also interrupted sequential NOE connectivities between nucleotides C5 and A6. The A6 deoxyribose ring showed an increased percentage of the C3'-endo conformation. This differed from the bay region BA RSRS (61,2) adduct, in which the lesion was located at position X6 [Li, Z., Mao, H., Kim, H.-Y., Tamura, P. J., Harris, C. M., Harris, T. M., and Stone, M. P. (1999) Biochemistry 38, 2969-2981], but was similar to the benzo[a]pyrene BP SRSR (61,3) adduct [Zegar I. S., Chary, P., Jabil, R. J., Tamura, P. J., Johansen, T. N., Lloyd, R. S., Harris, C. M., Harris, T. M., and Stone, M. P. (1998) Biochemistry 37, 16516-16528]. The altered sugar pseudorotation at A6 appears to be common to both bay region BA RSRS (61,3) and BP SRSR (61,3) adducts. It could not be discerned if the C3'-endo conformation at A6 in the BA RSRS (61,3) adduct altered base pairing geometry at X7 x T16, as compared to the C2'-endo conformation. The structural studies suggest that the mutational spectrum of this adduct may be more complex than that of the BA RSRS (61,2) adduct.     

Binding site of ribosomal proteins on prokaryotic 5S ribonucleic acids: a study with ribonucleases

The binding sites of ribosomal proteins L18 and L25 on 5S RNA from Escherichia coli were probed with ribonucleases A, T1, and T2 and a double helix specific cobra venom endonuclease. The results for the protein-RNA complexes, which were compared with those for the free RNA [Douthwaite, S., & Garrett, R. A. (1981) Biochemistry 20, 7301--7307], reveal an extensive interaction site for protein L18 and a more localized one for L25. Generally comparable results, with a few important differences, were obtained in a study of the binding sites of the two E. coli proteins on Bacillus stearothermophilus 5S RNA. Several protein-induced changes in the RNA structures were identified; some are possibly allosteric in nature. The two prokaryotic 5S RNAs were also incubated with total 50S subunit proteins from E. coli and B. stearothermophilus ribosomes. Homologous and heterologous reconstitution experiments were performed for both RNAs. The effects of the bound proteins on the ribonuclease digestion of the RNAs could generally be correlated with the results obtained with the E. coli proteins L18 and L25, although there was evidence for an additional protein-induced conformational change in the B. stearothermophilus 5S RNA, which may have been due to a third ribosomal protein L5.     

Structural characterization of a six-nucleotide RNA hairpin loop found in Escherichia coli, r(UUAAGU)

The binding region of the Escherichia coli S2 ribosomal protein contains a conserved UUAAGU hairpin loop. The structure of the hairpin formed by the oligomer r(GCGU4U5A6A7G8U9CGCA), which has an r(UUAAGU) hairpin loop, was determined by NMR and molecular modeling techniques as part of a study aimed at characterizing the structure and thermodynamics of RNA hairpin loops. Thermodynamic data obtained from melting curves for this RNA oligomer show that it forms a hairpin in solution with the following parameters: DeltaH degrees = -42.8 +/- 2.2 kcal/mol, DeltaS degrees = -127.6 +/- 6.5 eu, and DeltaG degrees (37) = -3.3 +/- 0.2 kcal/mol. Two-dimensional NOESY WATERGATE spectra show an NOE between U imino protons, which suggests that U4 and U9 form a hydrogen bonded U.U pair. The U5(H2') proton shows NOEs to both the A6(H8) proton and the A7(H8) proton, which is consistent with formation of a "U" turn between nucleotides U5 and A6. An NOE between the A7(H2) proton and the U9(H4') proton shows the proximity of the A7 base to the U9 sugar, which is consistent with the structure determined for the six-nucleotide loop. In addition to having a hydrogen-bonded U.U pair as the first mismatch and a U turn, the r(UUAAGU) loop has the G8 base protruding into the solvent. The solution structure of the r(UUAAGU) loop is essentially identical to the structure of an identical loop found in the crystal structure of the 30S ribosomal subunit where the guanine in the loop is involved in tertiary interactions with RNA bases from adjacent regions [Wimberly, B. T., Brodersen, D. E., Clemons, W. M., Morgan-Warren, R. J., Carter, A. P., Vonrhein, C., Hartsch, T., and Ramakrishnan, V. (2000) Nature 407, 327-339]. The similarity of the solution and solid-state structures of this hairpin loop suggests that formation of this hairpin may facilitate folding of 16S RNA.     

Silencing of RNA helicase II/Gualpha inhibits mammalian ribosomal RNA production

The intricate production of ribosomal RNA is well defined in yeast, but its complexity in higher organisms is barely understood. We recently showed that down-regulation of nucleolar protein RNA helicase II/Gualpha (RH-II/Gualpha or DDX21) in Xenopus oocytes inhibited processing of 20 S rRNA to 18 S and contributed to degradation of 28 S rRNA (Yang, H., Zhou, J., Ochs, R. L., Henning, D., Jin, R., and Valdez, B. C. (2003) J. Biol. Chem. 278, 38847-38859). Since no nucleolar RNA helicase has been functionally characterized in mammalian cells, we used short interfering RNA to search for functions for RH-II/Gualpha and its paralogue RH-II/Gubeta in rRNA production. Silencing of RH-II/Gualpha by more than 80% in HeLa cells resulted in an almost 80% inhibition of 18 and 28 S rRNA production. This inhibition could be reversed by exogenous expression of wild type RH-II/Gualpha. A helicase-deficient mutant form having ATPase activity was able to rescue the production of 28 S but not 18 S rRNA. A phenotype exhibiting inhibition of 18 S and 28 S rRNA production was also observed when the paralogue RH-II/Gubeta was overexpressed. Both down-regulation of RH-II/Gualpha and overexpression of RH-II/Gubeta slowed cell proliferation. The opposite effects of the two paralogues suggest antagonistic functions.     

Construction and functional analysis of ribosomal 5S RNA from Escherichia coli with single base changes in the ribosomal protein binding sites

The ribosomal 5S RNA gene from E. coli was altered by oligonucleotide-directed mutagenesis at positions A66 and U103. The mutant genes were cloned into an expression vector and selectively transcribed in an UV-sensitive E. coli strain using a modified maxicell system. The mutant 5S RNA genes were found to be transcribed and processed normally. The 5S RNA molecules were assembled into 50S ribosomal subunits. Under in vitro conditions the stability of the mutant 70S ribosomes seemed, however, to be reduced, since they dissociated into their subunits more easily than those of the wild type. The isolated mutated 5S RNAs with base changes in the ribosomal protein binding sites for L18 and L25, together with a point mutant at G41 (G to C), constructed earlier, were tested for their capacity to bind the 5S RNA binding proteins L5, L18 and L25. The following effects were observed: The base change A66 to C within the L18 binding site did not affect the binding of the ribosomal protein L18 but enhanced the stability of the L25-5S RNA complex considerably. The base changes U103 to G and G41 to C slightly reduced the binding of L5 and L25 whereas the binding of L18 to the mutant 5S RNAs was not altered. In addition 70S ribosomes with the single point mutations in their 5S RNAs were tested in their tRNA binding capacity. Mutants containing a C41 in their 5S RNA showed a reduction in the poly(U)-dependent Phe-tRNA binding, whereas the mutations to C66 and G 103 lead to completely inactive ribosomes in the same assay. Based on previous results a spatial model of the 5S RNA molecule is presented which is consistent with the findings reported in this paper.     

Phosphorothioate analogues of 2',5'-oligoadenylate. Enzymatic synthesis, properties, and biological activities of 2',5'-phosphorothioates from adenosine 5'-O-(2-thiotriphosphate) and adenosine 5'-O-(3-thiotriphosphate)

The chiral and achiral phosphorothioate analogues of 2',5'-oligoadenylates (2-5A) have been enzymatically synthesized from the Sp and Rp isomers of adenosine 5'-O-(2-thiotriphosphate) [(Sp)-ATP beta S and (Rp)-ATP beta S, respectively] and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) by 2-5A synthetase from L929 cells and lysed rabbit reticulocytes. These 2',5'-phosphorothioate analogues were separated, purified, and structurally characterized. While ATP gamma S and (Sp)-ATP beta S were as efficient substrates for the 2-5A synthetase as was ATP, (Rp)-ATP beta S was more than 50-fold less efficient a substrate. The beta- and gamma-phosphorothioates were more resistant to enzymatic hydrolysis than was authentic 2-5A. Compared to 2-5A, there were marked differences in the biological activities of the 2',5'-phosphorothioates as determined by (i) binding to 2-5A-dependent endoribonuclease (RNase L), (ii) activation of RNase L to hydrolyze RNA, and (iii) inhibition of protein synthesis in intact L929 cells. These studies extend previous reports on the elucidation of the stereochemical requirements of 2-5A synthetase and RNase L [Karikó, K., Sobol, R. W., Jr., Suhadolnik, L., Li, S. W., Reichenbach, N. L., Suhadolnik, R. J., Charubala, R., & Pfleiderer, W. (1987) Biochemistry (first of three papers in this issue); Karikó, K., Li, S. W., Sobol, R. W., Jr., Suhadolnik, R. J., Charubala, R., & Pfleiderer, W. (1987) Biochemistry (second of three papers in this issue)] with the phosphorothioate analogues of 2-5A.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR evidence for dynamic secondary structure in helices II and III of the RNA of Escherichia coli

A new ribonuclease A (RNase A) resistant fragment of the 5S ribonucleic acid (RNA) from Escherichia coli has been isolated and characterized. This fragment comprises helix III and most of helix II of the parent molecule, a part of the 5S RNA molecule for which several energetically equivalent secondary structures have been proposed [De Wachter, R., Chen, M.-W., & Vandenberghe, A. (1984) Eur. J. Biochem. 143, 175-182]. The imino proton spectrum of this fragment has been studied by nuclear magnetic resonance methods at 500 MHz. The data obtained are readily rationalized in terms of one of the structures proposed for this region of 5S RNA. They also suggest that upon heating, this structure is replaced by a second, different one, consistent with the view that the helix II-helix III region of 5S RNA is able to switch between alternative structures. Among the products of the nucleolytic digestion of 5S RNA is a species whose sequence indicates that RNase A can ligate RNA as well as hydrolyze it.     

Deoxyguanosine-deoxyadenosine pairing in the d(C-G-A-G-A-A-T-T-C-G-C-G) duplex: conformation and dynamics at and adjacent to the dG X dA mismatch site

Nuclear magnetic resonance (NMR) has been used to monitor the conformation and dynamics of the d-(C1-G2-A3-G4-A5-A6-T6-T5-C4-G3-C2-G1) self-complementary dodecanucleotide (henceforth called 12-mer GA) that contains a dG X dA purine-purine mismatch at position 3 in the sequence. These results are compared with the corresponding d(C-G-C-G-A-A-T-T-C-G-C-G) dodecamer duplex (henceforth called 12-mer) containing standard Watson-Crick base pairs at position 3 [Patel, D.J., Kozlowski, S.A., Marky, L.A., Broka, C., Rice, J.A., Itakura, K., & Breslauer, K.J. (1982) Biochemistry 21, 428-436]. The dG X dA interaction at position 3 was monitored at the guanosine exchangeable H-1 and nonexchangeable H-8 protons and the nonexchangeable adenosine H-2 proton. We demonstrate base-pair formation between anti orientations of the guanosine and adenosine rings on the basis of nuclear Overhauser effects (NOE) observed between the H-2 proton of adenosine 3 and the imino protons of guanosine 3 (intra base pair) and guanosines 2 and 4 (inter base pair). The dG(anti) X dA(anti) pairing should result in hydrogen-bond formation between the guanosine imino H-1 and carbonyl O-6 groups and the adenosine N-1 and NH2-6 groups, respectively. The base pairing on either side of the dG X dA pair remains intact at low temperature, but these dG X dC pairs at positions 2 and 4 are kinetically destabilized in the 12-mer GA compared to the 12-mer duplex. We have estimated the hydrogen exchange kinetics at positions 4-6 from saturation-recovery measurements on the imino protons of the 12-mer GA duplex between 5 and 40 degrees C. The measured activation energies for imino proton exchange in the 12-mer GA are larger by a factor of approximately 2 compared to the corresponding values in the 12-mer duplex. This implies that hydrogen exchange in the 12-mer GA duplex results from a cooperative transition involving exchange of several base pairs as was previously reported for the 12-mer containing a G X T wobble pair at position 3 [Pardi, A., Morden, K.M., Patel, D.J., & Tinoco, I., Jr. (1982) Biochemistry 21, 6567-6574]. We have assigned the nonexchangeable base protons by intra and inter base pair NOE experiments and monitored these assigned markers through the 12-mer GA duplex to strand transition.(ABSTRACT TRUNCATED AT 400 WORDS)     

The primary nucleotide sequence of U4 RNA

U4 RNA is one of the "capped" nuclear snRNAs recently found to be precipitable by anti-Sm antibodies as ribonucleoprotein particles. U4 RNA, along with other snRNAs, has been implicated in hnRNA processing, mRNA transport, or both (Lerner, M. R., Boyle, J., Mount, S., Wolin, S., and Steitz, J. A. (1980) Nature 283, 220-224). Since the proteins bound to different snRNAs appear to be the same, the functions of different snRNPs might be dependent on the RNA components. To help understand the function of U4 RNP, the nucleotide sequence of U4 RNA was determined. The sequence is (formula see text) In addition to the modified nucleotides in the "cap," U4 RNA contains Am at position 63 and m6A at position 98. It also exhibited A-C microheterogeneity at position 97.     

Recent advances in the molecular analysis of inherited disease

Many important human genes have been cloned during the last ten years. In some cases, using reverse genetic techniques [Orkin, S. H. (1986) Cell 47, 845-850], disease-causing genes have been isolated whose product was previously unknown. Important examples include the dystrophin protein which, when mutated, gives rise to either Duchenne or Becker muscular dystrophy [Koenig, M., Hoffman, E. P., Bertelson, C. J., Monaco, A. P., Feener, C. and Kunkel, L. M. (1987) Cell 50, 509-517; Monaco, A. P., Bertelson, C. J., Liechti-Gallati, S. & Kunkel, L. M. (1988) Genomics 2, 90-95; Koenig, M., Monaco, A. P. & Kunkel, L. M. (1988) Cell 53, 219-228] and the cystic fibrosis transmembrane conductance regulator (CFTR) [Riordan, J. R., Rommens, J. M., Kerem, B.-S., Alon, N., Rozmahel, R., Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., Chou, J.-L., Drumm, M. L., Ianuzzi, M. C., Collins, F. S. & Tsui, L.-C. (1989) Science 245, 1066-1073]. Recently the technology for systematically detecting single base-pair changes by chemical methods, enzymatic methods or direct DNA sequencing has greatly expanded and simplified. In addition to providing structural information about these clinically important genes and information on disease-causing mutations, these studies have led to an increased understanding of mechanisms of mutation, to the discovery of novel genetic mechanisms and to important clinical applications of carrier detection and pre-natal diagnosis. The recent rapid progress has been made possible by the development of DNA amplification using the polymerase chain reaction (pcr) invented by Saiki and colleagues [Saiki, R. K., Chang, C-A., Levenson, C. H., Warren, T. C., Boehm, C. D., Kazazian, H. H. & Ehrlich, H. A. (1988) N. Engl. J. Med. 319, 537-541].     

Arresting initiation of hepatitis C virus RNA synthesis using heterocyclic derivatives

The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRp), the primary catalytic enzyme of the HCV replicase complex. Recently, two benzo-1,2,4-thiadiazine compounds were shown to be potent, highly specific inhibitors of the genotype 1b HCV RdRp containing a carboxyl-terminal 21 residue truncation (delta21 HCV RdRp) (Dhanak, D., Duffy, K., Johnston, V. K., Lin-Goerke, J., Darcy, M., Shaw, A. N. G. B., Silverman, C., Gates, A. T., Earnshaw, D. L., Casper, D. J., Kaura, A., Baker, A., Greenwood, C., Gutshall, L. L., Maley, D., DelVecchio, A., Macarron, R., Hofmann, G. A., Alnoah, Z., Cheng, H.-Y., Chan, G., Khandekar, S., Keenan, R. M., and Sarisky, R. T. (2002) J. Biol. Chem. 277, 38322-38327). Compound 4 (C(21)H(21)N(3)O(4)S) reduces viral replication by virtue of its direct interaction with the viral polymerase rather than by nonspecific titration of nucleic acid template. In this study, we present several lines of evidence to demonstrate that this inhibitor interferes with the initiation step of RNA synthesis rather than acting as an elongation inhibitor. Inhibition of initial phosphodiester bond formation occurred regardless of whether replication was initiated by primer-dependent or de novo mechanisms. Filter binding studies using increasing concentrations of compound 4 did not interfere with the ability of delta21 HCV RdRp to interact with nucleic acid. Furthermore, varying the order of reagent addition in the primer extension assay showed no distinct differences in inhibition profile. Finally, surface plasmon resonance analyses provided evidence that a ternary complex is capable of forming between the RNA template, RdRp, and compound 4. Together, these data suggest that this heterocyclic agent interacts with the apoenzyme, as well as with the RNA-bound form of delta21 HCV RdRp, and therefore does not directly interfere with the RdRp-RNA interaction to mediate inhibition.     

Wheat germ poly(A)-binding protein increases the ATPase and the RNA helicase activity of translation initiation factors eIF4A, eIF4B, and eIF-iso4F

Recent studies demonstrated that wheat germ poly(A)-binding protein (PABP) interacted with translation eukaryotic initiation factor (eIF)-iso4G and eIF4B, and these interactions increased the poly(A) binding activity of PABP (Le, H., Tanguay, R. L., Balasta, M. L., Wei, C. C., Browning, K. S., Metz, A. M., Goss, D. J., and Gallie, D. R. (1997) J. Biol. Chem. 272, 16247-16255) and the cap binding activity of eIF-iso4F (Wei, C. C., Balasta, M. L., Ren, J., and Goss, D. J. (1998) Biochemistry 37, 1910-1916). We report here that the interaction between PABP and eIF-iso4G has a substantial effect on the ATPase activity and RNA helicase activity of (eIF4A + eIF4B + eIF-iso4F) complex. ATPase kinetic assays show, in the presence of poly(U), PABP can increase the parameter (k(cat)/K(m)) by 3.5-fold with a 2-fold decrease of K(m) for the (eIF4A + eIF-iso4F) complex. In the presence of globin messenger RNA, the ATPase activity of the complex (eIF4A + eIF-iso4F) was increased 2-fold by the presence of PABP. RNA helicase assays demonstrated that the presence of PABP enhanced the RNA duplex unwinding activity of the initiation factor complex. These results suggest that, in terms of the scanning model of translation initiation, PABP may enhance the mRNA scanning rate of the complex formed by eIF4A, eIF4B, and eIF4F or eIF-(iso)4F and increase the rate of translation.