Studies of point mutants define three essential paired nucleotides in the domain 5 substructure of a group II intron.

Domain 5 (D5) is a highly conserved, largely helical substructure of group II introns that is essential for self-splicing. Only three of the 14 base pairs present in most D5 structures (A2.U33, G3.U32, and C4.G31) are nearly invariant. We have studied effects of point mutations of those six nucleotides on self-splicing and in vivo splicing of aI5 gamma, an intron of the COXI gene of Saccharomyces cerevisiae mitochondria. Though none of the point mutations blocked self-splicing under one commonly used in vitro reaction condition, the most debilitating mutations were at G3 and G4. Following mitochondrial Biolistic transformation, it was found that mutations at A2, G3, and C4 blocked respiratory growth and splicing while mutations at the other sites had little effect on either phenotype. Intra-D5 second-site suppressors showed that pairing between nucleotides at positions 2 and 33 and 4 and 31 is especially important for D5 function. At the G3.U32 wobble pair, the mutant A.U pair blocks splicing, but a revertant of that mutant that can form an A+.C base pair regains some splicing. A dominant nuclear suppressor restores some splicing to the G3A mutant but not the G3U mutant, suggesting that a purine is required at position 3. These findings are discussed in terms of the hypothesis of Madhani and Guthrie (H. D. Madhani and C. Guthrie, Cell 71:803-817, 1992) that helix 1 formed between yeast U2 and U6 small nuclear RNAs may be the spliceosomal cognate of D5.     

The conserved U.G pair in the 5'' splice site duplex of a group I intron is required in the first but not the second step of self-splicing.

Group I self-splicing introns have a 5' splice site duplex (P1) that contains a single conserved base pair (U.G). The U is the last nucleotide of the 5' exon, and the G is part of the internal guide sequence within the intron. Using site-specific mutagenesis and analysis of the rate and accuracy of splicing of the Tetrahymena thermophila group I intron, we found that both the U and the G of the U.G pair are important for the first step of self-splicing (attack of GTP at the 5' splice site). Mutation of the U to a purine activated cryptic 5' splice sites in which a U.G pair was restored; this result emphasizes the preference for a U.G at the splice site. Nevertheless, some splicing persisted at the normal site after introduction of a purine, suggesting that position within the P1 helix is another determinant of 5' splice site choice. When the U was changed to a C, the accuracy of splicing was not affected, but the Km for GTP was increased by a factor of 15 and the catalytic rate constant was decreased by a factor of 7. Substitution of U.A, U.U, G.G, or A.G for the conserved U.G decreased the rate of splicing by an even greater amount. In contrast, mutation of the conserved G enhanced the second step of splicing, as evidenced by a trans-splicing assay. Furthermore, a free 5' exon ending in A or C instead of the conserved U underwent efficient ligation. Thus, unlike the remainder of the P1 helix, which functions in both the first and second steps of self-splicing, the conserved U.G appears to be important only for the first step.     

Divalent metal ion binding to a conserved wobble pair defining the upstream site of cleavage of group I self-splicing introns.

The upstream site of cleavage of all group I self-splicing introns is identified by an absolutely conserved U.G base pair. Although a wobble C.A pair can substitute the U.G pair, all other combinations of nucleotides at this position abolish splicing, suggesting that it is an unusual RNA structure, rather than sequence, that is recognized by the catalytic intron core. RNA enzymes are metalloenzymes, and divalent metal ion binding may be an important requirement for splice site recognition and catalysis. The paramagnetic broadening of NMR resonances upon manganese binding at specific sites was used to probe the interaction between divalent metal ions and an oligonucleotide model of a group I intron ribozyme substrate. Unlike previous studies in which only imino proton resonances were monitored, we have used isotopically labelled RNA and a set of complete spectral assignments to identify the location of the divalent metal binding site with much greater detail than previously possible. Two independent metal binding sites were identified for this oligonucleotide. A first metal binding site is located in the major groove of the three consecutive G.C base pairs at the end of double helical stem. A second site is found in the major groove of the RNA double helix in the vicinity of the U.G base pair. These results suggest that metal ion coordination (or a metal bridge) and tertiary interactions identified biochemically, may be used by group I intron ribozymes for substrate recognition.     

Role of the three consecutive G:C base pairs conserved in the anticodon stem of initiator tRNAs in initiation of protein synthesis in Escherichia coli.

The three consecutive G:C base pairs, G29:C41, G30:C40, and G31:C39, are conserved in the anticodon stem of virtually all initiator tRNAs from eubacteria, eukaryotes, and archaebacteria. We show that these G:C base pairs are important for function of the tRNA in initiation of protein synthesis in vivo. We changed these base pairs individually and in combinations and analyzed the activities of the mutant Escherichia coli initiator tRNAs in initiation in vivo. For assessment of activity of the mutant tRNAs in vivo, mutations in the G:C base pairs were coupled to mutation in the anticodon sequence from CAU to CUA. Mutations in each of the G:C base pairs reduced activity of the mutant tRNA in initiation, with mutation in the second G:C base pair having the most severe effect. The greatly reduced activity of this C30:G40 mutant tRNA is not due to defects in aminoacylation or formulation of the tRNA or defects in base modification of the A37, next to the anticodon, which we had previously shown to be important for activity of the mutant tRNAs in initiation. The anticodon stem mutants are most likely affected specifically at the step of binding to the ribosomal P site. The pattern of cleavages in the anticodon loop of mutant tRNAs by S1 nuclease indicate that the G:C base pairs may be involved directly in interactions of the tRNA with components of the P site on the ribosome rather than indirectly by inducing a particular conformation of the anticodon loop critical for function of the tRNA in initiation.     

The processing of wild type and mutant forms of rat nuclear pre-tRNA(Lys) by the homologous RNase P.

The 5' processing of rat pre-tRNA(Lys) and a series of mutant derivatives by rat cytosolic RNase P was examined. In standard, non-kinetic assays, mutant precursors synthesized in vitro with 5' leader sequences of 10, 17, 24, 25, and 46 nucleotides were processed to approximately equal levels and yielded precisely cleaved 5' processed intermediates with the normal 7-base pair aminoacyl stems. The construct containing the tRNA(Lys) with the 46-nucleotide leader was modified by PCR to give a series of pre-tRNA(Lys) mutants designed to measure the effect on processing by (1) substituting the nucleotide at the +1 position, (2) pairing and unpairing the +1 and +72 bases, (3) elongating the aminoacyl stem, and (4) disrupting the helix of the aminoacyl stem. Comparative kinetic analyses revealed that changing the wild type +1G to A, C, or U was well tolerated by the RNase P provided that compensatory changes at +72 created a base pair or a G.U noncanonical pair. Mutants with elongated aminoacyl stems that were produced either by inserting an additional base pair at +3:a + 69:a or by pairing the -1A with a +73U, were processed to yield 7-base pair aminoacyl stems, but with different efficiencies. The efficiency seen with the double insertion mutant was higher than even the wild type precursor, but the -1A-U + 73 mutant was a relatively poor substrate. Disrupting the aminoacyl stem helix by introducing a +7G G + 66 mispairing or by inserting a single G at the +3:a position dramatically reduced the processing efficiency, although the position of cleavage occurred precisely at the wild type cleavage site. However, the single insertion of a C at the +69:a position resulted in an efficiently cleaved precursor, but permitted a minor, secondary cleavage within the leader between the -6 and -5 nucleotides in addition to the dominant wild type scission.     

Anion binding to the Schiff base of the bacteriorhodopsin mutants Asp-85----Asn/Asp-212----Asn and Arg-82----Gln/Asp-85----Asn/Asp-212----Asn.

Studies of bacteriorhodopsin have indicated that the charge environment of the protonated Schiff base consists of residues Asp-85, Asp-212, and Arg-82. As shown recently (Marti, T., R?sselet, S. J., Otto, H., Heyn, M. P., and Khorana, H. G. (1991) J. Biol. Chem. 266, 18674-18683), in the double mutant Asp-85----Asn/Asp-212----Asn chromophore formation is restored in the presence of salts, suggesting that exogenous anions function as counterions to the protonated Schiff base. To investigate the role of Arg-82 and of the Schiff base in anion binding, we have prepared the triple mutant Arg-82----Gln/Asp-85----Asn/Asp-212----Asn and compared its properties with those of the Asp-85----Asn/Asp-212----Asn double mutant. Regeneration of the chromophore with absorption maximum near 560 nm occurs in the triple mutant in the presence of millimolar salt, whereas in the double mutant molar salt concentrations are required. Spectrometric titrations reveal that the pKa of Schiff base deprotonation is markedly reduced from 11.3 for the wild type to 4.9 for the triple mutant in 1 mM NaCl and to 5.5 for the double mutant in 10 mM NaCl. In both mutants, increasing the chloride concentration promotes protonation of the chromophore and results in a continuous rise of the Schiff base pKa, yielding a value of 8.4 and 7.6, respectively, in 4 M NaCl. The absorption maximum of the two mutants shows a progressive red shift, as the ionic radius of the halide increases in the sequence fluoride, chloride, bromide, and iodide. An identical spectral correlation in the presence of halides is observed for the acid-purple form of bacteriorhodopsin. We conclude, therefore, that upon neutralization of the two counterions Asp-85 and Asp-212 by mutation or by protonation at low pH, exogenous anions substitute as counterions by directly binding to the protonated Schiff base. This interaction may provide the basis for the proposed anion translocation by the acid-purple form of bacteriorhodopsin as well as by the related halorhodopsin.     

Mutational analysis of the RNA pseudoknot involved in efficient ribosomal frameshifting in simian retrovirus-1.

Mutational effects on frameshifting efficiency of the RNA pseudoknot involved in ribosomal frameshifting in simian retrovirus-1 (SRV-1) have been investigated. The primary sequence and the proposed secondary structure of the SRV-1 pseudoknot are similar to those of other efficient frameshifting pseudoknots in mouse mammary tumor virus (MMTV) and feline immunodeficiency virus (FIV), where an unpaired adenine nucleotide intercalates between stem 1 and stem 2. In SRV-1 pseudoknot, the adenine nucleotide in between stem 1 and stem 2 has a potential to form an A*U base pair with the last uridine nucleotide in the loop 2, resulting in a continuous A-form helix with coaxially stacked stem 1 and stem 2. To test whether this A*U base pairing and coaxial stacking of stem 1 and stem 2 is absolutely required for efficient frameshifting in SRV-1, a series of mutants changing this potential A.U base pair to either G.C base pair or A.A, A.G, A.C, G.A, G.G mismatch is generated, and their frameshifting efficiencies are investigated in vitro using rabbit reticulocyte lysate translation assay. The frameshifting abilities of these mutant pseudoknots are similar to that of the wild-type pseudoknot, suggesting that the A*U base pair in between stem 1 and stem 2 is not necessary to promote efficient frameshifting in SRV-1. These results reveal that coaxial stacking of stem 1 and stem 2 with a Watson-Crick A.U base pair in between two stems is not a required structural feature of the pseudoknot for promoting efficient frameshifting in SRV-1. Our mutational data suggest that SRV-1 pseudoknot adopts similar structural features common to other efficient frameshifting pseudoknots as observed in MMTV and FIV.     

Mutations in the anticodon stem affect removal of introns from pre-tRNA in Saccharomyces cerevisiae.

To evaluate the role of exon domains in tRNA splicing, the anti-codon stem of proline pre-tRNAUGG from Saccharomyces cerevisiae was altered by site-directed mutagenesis of the suf8 gene. Sixteen alleles were constructed that encode mutant pre-tRNAs containing all possible base combinations in the last base pair of the anticodon stem adjacent to the anticodon loop (positions 31 and 39). The altered pre-tRNAs were screened by using an in vitro endonucleolytic cleavage assay to determine whether perturbations in secondary structure affect the intron excision reaction. The pre-tRNAs were cleaved efficiently whenever secondary structure in the anticodon stem was maintained through standard base pairing or G.U interactions. However, most of the pre-tRNAs with disrupted secondary structure were poor substrates for intron excision. We also determined the extent to which the suf8 alleles produce functional products in vivo. Each allele was integrated in one to three copies into a yeast chromosome or introduced on a high-copy-number plasmid by transformation. The formation of a functional product was assayed by the ability of each allele to suppress the +1 frameshift mutation his4-713 through four-base codon reading, as shown previously for the SUF8-1 suppressor allele. We found that alleles containing any standard base pair or G.U pair at position 31/39 in the anticodon stem failed to suppress his4-713. We could not assess in vivo splicing with these alleles because the tRNA products, even if they are made, would be expected to read a normal triplet rather than a quadruplet codon. However, all of the alleles that contained a disrupted base pair at position 31/ 39 in the anticodon stem altered the structure of the tRNA in a manner that caused frameshift suppression. Suppression indicated that splicing must have occurred to some extent in vivo even though most of the suppression alleles produced pre-tRNAs that were cleaved with low efficiency or not at all in vitro. These results have important implications for the interpretation of in vitro cleavage assays in general and for the potential use of suppressors to select mutations that affects tRNA splicing.     

Minimal catalytic domain of a group I self-splicing intron RNA

The self-splicing intron ribozymes have been regarded as primitive forms of the splicing machinery for eukaryotic pre-mRNAs. The splicing activity of group I self-splicing introns is dependent on an absolutely conserved and exceptionally densely packed core region composed of two helical domains, P3-P7 and P4-P6, that are connected rigidly via base triples. Here we show that a mutant group I intron ribozyme lacking both the P4-P6 domain and the base triples can perform the phosphoester transfer reactions required for splicing at both the 5' and 3' splice sites, demonstrating that the elements required for splicing are concentrated in the stacked helical P3-P7 domain. This finding establishes that the conserved core of the intron consists of two physically and functionally separable components, and we present a model showing the architecture of a prototype of this class of intron and the course of its molecular evolution.     

Probing the role of a secondary structure element at the 5'- and 3'-splice sites in group I intron self-splicing: the tetrahymena L-16 ScaI ribozyme reveals a new role of the G.U pair in self-splicing

Several ribozyme constructs have been used to dissect aspects of the group I self-splicing reaction. The Tetrahymena L-21 ScaI ribozyme, the best studied of these intron analogues, catalyzes a reaction analogous to the first step of self-splicing, in which a 5'-splice site analogue (S) and guanosine (G) are converted into a 5'-exon analogue (P) and GA. This ribozyme preserves the active site but lacks a short 5'-terminal segment (called the IGS extension herein) that forms dynamic helices, called the P1 extension and P10 helix. The P1 extension forms at the 5'-splice site in the first step of self-splicing, and P10 forms at the 3'-splice site in the second step of self-splicing. To dissect the contributions from the IGS extension and the helices it forms, we have investigated the effects of each of these elements at each reaction step. These experiments were performed with the L-16 ScaI ribozyme, which retains the IGS extension, and with 5'- and 3'-splice site analogues that differ in their ability to form the helices. The presence of the IGS extension strengthens binding of P by 40-fold, even when no new base pairs are formed. This large effect was especially surprising, as binding of S is essentially unaffected for S analogues that do not form additional base pairs with the IGS extension. Analysis of a U.U pair immediately 3' to the cleavage site suggests that a previously identified deleterious effect from a dangling U residue on the L-21 ScaI ribozyme arises from a fortuitous active site interaction and has implications for RNA tertiary structure specificity. Comparisons of the affinities of 5'-splice site analogues that form only a subset of base pairs reveal that inclusion of the conserved G.U base pair at the cleavage site of group I introns destabilizes the P1 extension >100-fold relative to the stability of a helix with all Watson-Crick base pairs. Previous structural data with model duplexes and the recent intron structures suggest that this effect can be attributed to partial unstacking of the P1 extension at the G.U step. These results suggest a previously unrecognized role of the G.U wobble pair in self-splicing: breaking cooperativity in base pair formation between P1 and the P1 extensions. This effect may facilitate replacement of the P1 extension with P10 after the first chemical step of self-splicing and release of the ligated exons after the second step of self-splicing.     

Mutagenesis and comparative sequence analysis of a base triple joining the two domains of group I ribozymes.

Tertiary interactions are important in the higher-order folding of catalytic RNAs. Recently, a base triple, joining the two major domains of the catalytic core, was determined in group I introns from the cyanobacterium Anabaena PCC7120 and the eukaryote Tetrahymena thermophila. This base triple involves the fifth base pair of P4 and the fifth base of the single-stranded region J8/7. We made base pair and single-nucleotide substitutions in the fifth base pair of P4, a G-C in the wild-type Anabaena intron, and tested them for self-splicing activity. The results suggest a hydrogen bonding model in which only the C of the base pair interacts directly with the fifth base of J8/7. Comparative sequence analysis was used to determine the different combinations of base triples that occur in approximately 450 natural group I introns identified to date. About 94% of the base triples analyzed are compatible with the proposed hydrogen bonding model. Disrupting this base triple in the Tetrahymena intron resulted in the disappearance of splicing intermediates (intron 3' exon and 5' exon), even though the first step of splicing was not affected. Restoration of the base triple by a compensatory mutation reverted the intermediates to wild-type levels. These results suggest that disruption of the base triple increases the rate of the second step of splicing or of a conformational change preceding the second step. Repositioning of the base triple to form a new set of interactions may be required for the second step of splicing.     

Structural analysis of the Neurospora mitochondrial large rRNA intron and construction of a mini-intron that shows protein-dependent splicing.

The gene encoding the Neurospora mitochondrial large rRNA contains a single group I intron of 2.3 kilobases that is not self-splicing in vitro. We showed previously that the splicing of this intron in vivo and in vitro is dependent on the Neurospora cyt-18 protein, mitochondrial tyrosyl-tRNA synthetase. In the present work, we carried out further structural analysis of the intron and constructed mutant derivatives of it in order to identify features that are either required for splicing or prevent it from self-splicing. Previous studies showed that the intron contains a large hairpin structure near the 5' splice site. By mapping RNase III cleavage sites, we identified this hairpin structure as an extended P2 stem. We construct a mini-intron of 388 nucleotides by deleting the 426-amino acid intron open reading frame, most of the 5' intron hairpin, and all of L8. This mini-intron shows the same protein-dependent splicing as the full length intron, but is still not self-splicing. Further deletions, which remove all of P2 or all or part of P4, P6, P7, or P9, inactivate splicing, suggesting that an intact group I intron core structure is required. Strengthening the P1, P10, or P9.0 pairings did not enable the mini-intron to self-splice. Our findings indicate that the inability of the mitochondrial large rRNA intron to self-splice reflects deficiency of a structure or activity required for cleavage at the 5' splice site, either in the intron core itself or in the interaction between the core and the P1 stem.     

Group II intron splicing in chloroplasts: identificationof mutations determining intron stability and fate of exon RNA.

In order to investigate in vivo splicing of group II introns in chloroplasts, we previously have integrated the mitochondrial intron rI1 from the green alga Scenedesmus obliquus into the Chlamydomonas chloroplast tscA gene. This construct allows a functional analysis of conserved intron sequences in vivo, since intron rI1 is correctly spliced in chloroplasts. Using site-directed mutagenesis, deletions of the conserved intron domains V and VI were performed. In another set of experiments, each possible substitution of the strictly conserved first intron nucleotide G1 was generated, as well as each possible single and double mutation of the tertiary base pairing gamma-gamma ' involved in the formation of the intron's tertiary RNA structure. In most cases, the intron mutations showed the same effect on in vivo intron splicing efficiency as they did on the in vitro self-splicing reaction, since catalytic activity is provided by the intron RNA itself. In vivo, all mutations have additional effects on the chimeric tscA -rI1 RNA, most probably due to the role played by trans -acting factors in intron processing. Substitutions of the gamma-gamma ' base pair lead to an accumulation of excised intron RNA, since intron stability is increased. In sharp contrast to autocatalytic splicing, all point mutations result in a complete loss of exon RNA, although the spliced intron accumulates to high levels. Intron degradation and exon ligation only occur in double mutants with restored base pairing between the gamma and gamma' sites. Therefore, we conclude that intron degradation, as well as the ligation of exon-exon molecules, depends on the tertiary intron structure. Furthermore, our data suggest that intron excision proceeds in vivo independent of ligation of exon-exon molecules.     

Mutations at the 3' splice site can be suppressed by compensatory base changes in U1 snRNA in fission yeast.

U1 snRNA is an essential splicing factor known to base pair with 5' splice sites of premessenger RNAs. We demonstrate that pairing between the universally conserved CU just downstream from the 5' junction interaction region and the 3' splice site AG contributes to efficient splicing of Schizosaccharomyces pombe introns that typify the AG-dependent class described in mammals. Strains carrying mutations in the 3' AG of an artificial intron accumulate linear precursor, indicative of a first step block. Lariat formation is partially restored in these mutants by compensatory changes in nucleotides C7 and U8 of U1 snRNA. Consistent with a general role in fission yeast splicing, mutations at C7 are lethal, while U8 mutants are growth impaired and accumulate linear, unspliced precursor to U6 snRNA. U1 RNA-mediated recognition of the 3' splice site may have origins in analogous intramolecular interactions in an ancestral self-splicing RNA.     

Dynamics in the U6 RNA intramolecular stem-loop: a base flipping conformational change

The U6 RNA intramolecular stem-loop (ISL) structure is an essential component of the spliceosome and binds a metal ion required for pre-messenger RNA splicing. The metal binding internal loop region of the stem contains a partially protonated C67-(+)A79 base pair (pK(a) = 6.5) and an unpaired U80 nucleotide that is stacked within the helix at pH 7.0. Here, we determine that protonation occurs with an exchange lifetime of approximately 20 micros and report the solution structures of the U6 ISL at pH 5.7. The differences between pH 5.7 and 7.0 structures reveal that the pH change significantly alters the RNA conformation. At lower pH, U80 is flipped out into the major groove. Base flipping involves a purine stacking interaction of flanking nucleotides, inversion of the sugar pucker 5' to the flipped base, and phosphodiester backbone rearrangement. Analysis of residual dipolar couplings as a function of pH indicates that base flipping is not restricted to a local conformational change. Rather, base flipping alters the alignment of the upper and lower helices. The alternative conformations of the U6 ISL reveal striking structural similarities with both the NMR and crystal structures of domain 5 of self-splicing group II introns. These structures suggest that base flipping at an essential metal binding site is a conserved feature of the splicing machinery for both the spliceosome and group II self-splicing introns.     

A non-directed, hydroxylamine-generated suppressor mutation in the P3 pairing region of the bacteriophage T4 td intron partially restores self-splicing capability

Hydroxylamine (HA) mutagenesis of an HA-induced splicing-defective bacteriophage T4 td intron mutant with a mutation in the intron P3 RNA pairing region was used to generate pseudorevertants. Because HA can only cause GC to AT transitions, the original mutant (H104A) could not undergo true reversion, yet the compensatory mutation on the opposite side of the P3 helix, which was complementary to the original H104A mutation, could occur. A pseudorevertant was isolated that contained both the original H104A mutation and the compensatory mutation HS9. By phenotypic and molecular genetic criteria, this double mutant (H104A-HS9) was shown to be able to undergo significant RNA splicing, thus confirming the existence and functional importance of the long-range P3 pairing region in this phage intron. The second-site suppressor mutation (HS9) was isolated by phage cross and also exhibited some self-splicing ability. A correlation exists between the strength of P3 helix Watson-Crick base pairing and the apparent level of splicing when wild-type, H104A, HS9, and H104A-HS9 are compared. This suggests that the primary role of the P3 RNA pairing region in the T4 td intron is structural in contributing to the critical RNA secondary structure.     

A dynamic bulge in the U6 RNA internal stem-loop functions in spliceosome assembly and activation

The highly conserved internal stem-loop (ISL) of U6 spliceosomal RNA is unwound for U4/U6 complex formation during spliceosome assembly and reformed upon U4 release during spliceosome activation. The U6 ISL is structurally similar to Domain 5 of group II self-splicing introns, and contains a dynamic bulge that coordinates a Mg++ ion essential for the first catalytic step of splicing. We have analyzed the causes of growth defects resulting from mutations in the Saccharomyces cerevisiae U6 ISL-bulged nucleotide U80 and the adjacent C67-A79 base pair. Intragenic suppressors and enhancers of the cold-sensitive A79G mutation, which replaces the C-A pair with a C-G pair, suggest that it stabilizes the ISL, inhibits U4/U6 assembly, and may also disrupt spliceosome activation. The lethality of mutations C67A and C67G results from disruption of base-pairing potential between U4 and U6, as these mutations are fully suppressed by compensatory mutations in U4 RNA. Strikingly, suppressor analysis shows that the lethality of the U80G mutation is due not only to formation of a stable base pair with C67, as previously proposed, but also another defect. A U6-U80G strain in which mispairing with position 67 is prevented grows poorly and assembles aberrant spliceosomes that retain U1 snRNP and fail to fully unwind the U4/U6 complex at elevated temperatures. Our data suggest that the U6 ISL bulge is important for coupling U1 snRNP release with U4/U6 unwinding during spliceosome activation.     

Human endonuclease III acts preferentially on DNA damage opposite guanine residues in DNA

The human endonuclease III homologue (hNTH1) removes premutagenic cytosine damage from DNA. This includes 5-hydroxycytosine, which has increased potential for pairing with adenine, resulting in C --> T transition mutations. Here we report that hNTH1 acts on both 5-hydroxycytosine and abasic sites preferentially when these are situated opposite guanines in DNA. Discrimination against other opposite bases is strongly dependent on the presence of magnesium. To further elucidate this effect, we have introduced mutations in the helix-hairpin-helix domain of hNTH1 (K212S, P211R, +G212, and DeltaP211), and measured the kinetics of 5-hydroxycytosine removal of the mutants relative to wild type. The K212S and DeltaP211 (truncated hairpin) mutant proteins were both inactive, whereas the extended hairpin in the +G212 mutant diminished recognition and binding to 5-hydroxycytosine-containing DNA. The P211R mutant resembled native hNTH1, except for decreased specificity of binding. Despite the altered kinetic parameters, the active mutants retained the ability to discriminate against the pairing base, indicating that enzyme interactions with the opposite strand relies on other domains than the active site helix-hairpin-helix motif.     

Vibrational spectroscopy of bacteriorhodopsin mutants. Evidence for the interaction of aspartic acid 212 with tyrosine 185 and possible role in the proton pump mechanism

The role of Asp-212 in the proton pumping mechanism of bacteriorhodopsin (bR) has been studied by a combination of site-directed mutagenesis and Fourier transform infrared difference spectroscopy. Difference spectra were recorded at low temperature for the bR----K and bR----M photoreactions of the mutants Asp-212----Glu, Asp-212----Asn, and Asp-212----Ala. Despite an increased proportion of the 13-cis form of bR (normally associated with dark adaptation), all of the mutants exhibited a light-adapted form containing as a principal component the normal all-trans retinal chromophore. The absence of a shift in the retinal C = C stretching frequency in these mutants indicates that Asp-212 is not a major determinant of the visible absorption wavelength maximum in light-adapted bR. It is unlikely that Asp-212 is the acceptor group for the Schiff base proton since both the Asp-212----Glu and Asp-212----Ala mutants formed an M intermediate. All of the Asp-212 mutants were missing a Fourier transform infrared difference band that had been assigned previously to protonation changes of Tyr-185. These results are discussed in terms of a model in which Tyr-185 and Asp-212 form a polarizable hydrogen bond and are positioned near the C13-Schiff base portion of the chromophore. These 2 residues may be involved in stabilizing the relative orientation of the F and G helices and isomerizing the retinal in a regioselective manner about the C13 = C14 double bond.