We determined the crystal structure of a bifunctional group I intron splicing factor and homing endonuclease, termed the I-AniI maturase, in complex with its DNA target at 2.6 Å resolution. The structure demonstrates the remarkable structural conservation of the -sheet DNA-binding motif between highly divergent enzyme subfamilies. DNA recognition by I-AniI was further studied using nucleoside deletion and DMS modification interference analyses. Correlation of these results with the crystal structure provides information on the relative importance of individual nucleotide contacts for DNA recognition. Alignment and modeling of two homologous maturases reveals conserved basic surface residues, distant from the DNA-binding surface, that might be involved in RNA binding. A point mutation that introduces a single negative charge in this region uncouples the maturase and endonuclease functions of the protein, inhibiting RNA binding and splicing while maintaining DNA binding and cleavage.
We have identified four mutations in Xenopus TFIIIA that increase the stability of TFIIIA-5 S rRNA gene complexes. In each case, the mutation has a relatively modest effect on equilibrium binding affinity. In three cases, these equilibrium binding effects can be ascribed primarily to decreases in the rate constant for protein-DNA complex dissociation. In the fourth case, however, a substitution of phenylalanine for the wild-type leucine at position 148 in TFIIIA results in much larger compensating changes in the kinetics of complex assembly and dissociation. The data support a model in which a relatively unstable population of complexes with multi-component dissociation kinetics forms rapidly; complexes then undergo a slow conformational change that results in very stable, kinetically homogeneous TFIIIA-DNA complexes. The L148F mutant protein acts as a particularly potent transcriptional activator when it is fused to the VP16 activation domain and expressed in yeast cells. Substitution of L148 to tyrosine or tryptophan produces an equally strong transcriptional activator. Substitution to histidine results in genetic and biochemical effects that are more modest than, but similar to, those observed with the L148F mutation. We propose that an amino acid with a planar side chain at position 148 can intercalate between adjacent base pairs in the intermediate element of the 5 S rRNA gene. Intercalation occurs slowly but results in a very stable DNA-protein complex. These results suggest that transcriptional activation by a cis-acting sequence element is largely dependent on the kinetic, rather than the thermodynamic, stability of the complex formed with an activator protein. Thus, transcriptional activation is dependent in large part on the lifetime of the activator-DNA complex rather than on binding site occupancy at steady state. Introduction of intercalating amino acids into zinc finger proteins may be a useful tool for producing artificial transcription factors with particularly high in vivo activity.TFIIIA 1 (transcription factor IIIA) is both representative of the large family of zinc finger proteins encoded in eukaryotic genomes (1, 2) and unusually interesting among these proteins in several respects. It has the atypical ability to bind with high affinity and specificity to both DNA (3-6) and RNA (7-9), has an unusually large number of zinc finger domains (nine), and an extended DNA recognition sequence (the ϳ52-base pair internal control region of 5 S rRNA genes), exhibits rather complex binding energetics to both DNA (10 -12) and RNA (13), and is remarkably poorly conserved during the evolution of eukaryotes (14, 15).We have described previously a method for the genetic analysis of the Xenopus TFIIIA-5 S rRNA gene interaction (16,17). In this approach, the Xenopus 5 S rRNA gene substitutes for the natural upstream activating sequence in the Saccharomyces cerevisiae CYC1 promoter which, in turn, controls expression of the Escherichia coli -galactosidase (lacZ) gene in yeast cells. Transcriptional activity fr...
Group I introns often encode proteins that catalyze site-specific DNA hydrolysis. Some of these proteins have acquired the ability to promote splicing of their cognate intron, but whether these two activities reside in different regions of the protein remains obscure. A crystal structure of I-AniI, a dual function intron-encoded protein, has shown that the protein has two pseudo-symmetric domains of equal size. Each domain contacts its DNA substrate on either side of two cleavage sites. As a first step to identify the RNA binding surface, the N-and C-terminal domains of I-AniI were separately expressed and tested for promoting the splicing of the mitochondrial (mt) COB pre-RNA. The N-terminal protein showed no splicing activation or RNA binding, suggesting that this domain plays a minimal role in activity or is improperly folded. Remarkably, the 16-kDa C-terminal half facilitates intron splicing with a rate similar to that of the full-length protein. Both the C-terminal fragment and full-length proteins bind tightly to the COB intron. RNase footprinting shows that the C-terminal and full-length proteins bind to the same regions and induce the same conformational changes in the COB intron. Together, these results show that the C-terminal fragment of I-AniI is necessary and sufficient for maturase activity and suggests that I-AniI acquired splicing function by utilizing a relatively small protein surface that likely represents a novel RNA binding motif. This fragment of I-AniI represents the smallest group I intron splicing cofactor described to date.
) have proposed that several amino acid side chains exhibit considerable conformational mobility at the DNA-protein interface in the transcription factor IIIA⅐5 S rRNA gene complex and that the rapid movements of these side chains permit them to make fluctuating contacts with adjacent bp in the DNA target site. This "dynamic interface" model makes biochemical predictions concerning the consequences of truncating specific amino acid side chains and the effects of these truncations on sequence selectivity in DNA binding. The model also makes predictions concerning the effects of DNA sequence context on the apparent energetic contributions to binding made by individual bp. We have tested these predictions, and our results are inconsistent with any significant energetic role being played by the contact of multiple bp by conformationally mobile amino acid side chains. They do, however, show that some individual amino acids affect the recognition of multiple bp through mechanisms other than direct interaction.Transcription factor IIIA (TFIIIA) 1 from Xenopus laevis exhibits a number of unusual features that make it of special interest in the study of nucleic acid-protein interactions. These include its ability to recognize both DNA (the internal control region of the 5 S rRNA gene) and RNA (5 S rRNA) with high affinity and specificity (1-4), its unusually large number of zinc fingers (nine), and correspondingly large DNA binding site (5-8), the complex thermodynamics with which it recognizes both DNA (9, 10) and RNA (11, 12), and its status as the archetypal zinc finger protein (13). Although it has been the subject of numerous biochemical studies, direct determination of the structure of the TFIIIA⅐5 S rRNA gene complex has been limited to two TFIIIA fragments bound to portions of the 5 S rRNA gene internal control region. Wright and colleagues (14, 15) have used nuclear magnetic resonance methods to determine the structure of the first three zinc fingers of TFIIIA bound to a 13-bp fragment of the 5 S rRNA gene, and Nolte et al. (16) have described a structure for the first six zinc fingers of TFIIIA bound to a 32-bp fragment of the same gene in a DNA-protein co-crystal. The structural models resulting from these studies confirmed the existence of some DNA-protein interactions in the TFIIIA⅐5 S rRNA gene complex that were predicted from the previously determined structures of other zinc finger proteins of the TFIIIA class (17-20) and also revealed previously undescribed features of DNA recognition by a zinc finger protein.Among the most interesting novel features of recognition proposed by Wright and colleagues was the existence of a dynamic DNA-protein interface in the complex of the three N-terminal zinc fingers of TFIIIA bound to bp 80 -92 in the 5 S rRNA gene. In particular, Foster et al. (15) and Wuttke et al. (14) proposed that several amino acid side chains involved in direct contact with DNA bp in the complex undergo rapid conformational fluctuations (in the s-ms time range). Furthermore, the authors propo...
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