What is the structure of the RecA-DNA filament?

The bacterial RecA protein has been a model system for understanding how a protein can catalyze homologous genetic recombination. RecA-like proteins have now been characterized from many organisms, from bacteriophage to humans. Some of the RecA-like proteins, including human RAD51, appear to function as helical filaments formed on DNA. However, we currently have high resolution structures of inactive forms of the protein, and low resolution structures of the active complexes formed by RecA-like proteins on DNA in the presence of ATP or ATP analogs. Within a crystal of the E. coli RecA protein, a helical polymer exists, and it has been widely assumed that this polymer is quite similar to the active helical filament formed on DNA. Recent developments have suggested that this may not be the case.

Comparison of bacteriophage T4 UvsX and human Rad51 filaments suggests that RecA-like polymers may have evolved independently.

The UvsX protein from bacteriophage T4 is a member of the RecA/Rad51/RadA family of recombinases active in homologous genetic recombination. Like RecA, Rad51 and RadA, UvsX forms helical filaments on DNA. We have used electron microscopy and a novel method for image analysis of helical filaments to show that UvsX-DNA filaments exist in two different conformations: an ADP state and an ATP state. As with RecA protein, these two states have a large difference in pitch. Remarkably, even though UvsX is only weakly homologous to RecA, both UvsX filament states are more similar to the RecA crystal structure than are RecA-DNA filaments. We use this similarity to fit the RecA crystal structure into the UvsX filament, and show that two of the three previously described blocks of similarity between UvsX and RecA are involved in the subunit-subunit interface in both the UvsX filament and the RecA crystal filament. Conversely, we show that human Rad51-DNA filaments have a different subunit-subunit interface than is present in the RecA crystal, and this interface involves two blocks of sequence similarity between Rad51 and RecA that do not overlap with those found between UvsX and RecA. This suggests that helical filaments in the RecA/Rad51/RadA family may have arisen from convergent evolution, with a conserved core structure that has assembled into multimeric filaments in a number of different ways.

The primase active site is on the outside of the hexameric bacteriophage T7 gene 4 helicase-primase ring.

Gene 4 of bacteriophage T7 encodes a protein (gp4) that can translocate along single-stranded DNA, couple the unwinding of duplex DNA with the hydrolysis of dTTP, and catalyze the synthesis of short RNA oligoribonucleotides for use as primers by T7 DNA polymerase. Electron microscopic studies have shown that gp4 forms hexameric rings, and X-ray crystal structures of the gp4 helicase domain and of the highly homologous RNA polymerase domain of Escherichia coli DnaG have been determined. Earlier biochemical studies have shown that when single-stranded DNA is bound to the hexameric ring, the primase domain remains accessible to free DNA. Given these results, a model was suggested in which the primase active site in the gp4 hexamer is located on the outside of the hexameric ring. We have used electron microscopy and single-particle image analysis to examine T7 gp4, and have determined that the primase active site is located on the outside of the hexameric ring, and therefore provide direct structural support for this model.

Electron microscopic studies of the translin octameric ring.

Translin is thought to participate in a variety of cellular activities including chromosomal translocations, translational regulation of mRNA expression, and mRNA transport. It forms an octameric ring structure capable of sequence-specific binding of both DNA and RNA substrates. We have used electron microscopy and single-particle image analysis to generate a three-dimensional reconstruction of the Translin ring. The subunits appear to have two distinct domains that assemble to form an open channel with diameter of approximately 30 A at one end and approximately 50 A at the opposite end. In the presence of either DNA or RNA containing consensus binding sequences, the largest opening into the central cavity is filled with density. Strikingly, although Translin shows significant sequence homology to only one other protein, Translin-associated factor X, the quaternary organization and the dimerization of subunits in the ring are very similar to those observed for hexameric ring helicases. This suggests that many of the structures in DNA and RNA metabolism may have similar quaternary organization.

Probing the structure of F-actin: cross-links constrain atomic models and modify actin dynamics.

Cross-links between protomers in F-actin can be used as a very sensitive probe of both the dynamics and structure of F-actin. We have characterized filaments formed from a previously described yeast actin Q41C mutant, where disulfide bonds can be formed between the Cys41 that is introduced into subdomain-2 and Cys374 on an adjacent protomer. We find that the distribution of cross-linked n-mers shows no cooperativity and corresponds to a random probability cross-linking reaction. The random distribution suggests that disulfide formation does not cause a significant perturbation of the F-actin structure. Consistent with this lack of perturbation, three-dimensional reconstructions of extensively cross-linked filaments, using a new approach to helical image analysis, show very small structural changes with respect to uncross-linked filaments. This finding is in conflict with refined models but in agreement with the original Holmes et al. model for F-actin. Under conditions where 94 % of the protomers are linked by disulfide bonds, the distribution of filament twist becomes more heterogeneous with respect to control filaments. A molecular model suggests that strain, introduced by the disulfide, is relieved by increasing the twist of the long-pitch actin helices. Disulfide formation makes yeast actin filaments approximately three times less flexible in terms of bending and similar, in this respect, to vertebrate skeletal muscle F-actin. These observations support previous reports that the rigidity of F-actin can be controlled by the position of subdomain-2, and that this region is more flexible in yeast F-actin than in skeletal muscle F-actin.

Movement of the decoding region of the 16 S ribosomal RNA accompanies tRNA translocation.

The ribosome undergoes pronounced periodic conformational changes during protein synthesis. Of particular importance are those occurring around the decoding site, the region of the 16 S rRNA interacting with the mRNA-(tRNA)(2) complex. We have incorporated structural information from X-ray crystallography and nuclear magnetic resonance into cryo-electron microscopic maps of ribosomal complexes designed to capture structural changes at the translocation step of the polypeptide elongation cycle. The A-site region of the decoding site actively participates in the translocation of the tRNA from the A to the P-site upon GTP hydrolysis by elongation factor G, shifting approximately 8 A toward the P-site. This implies that elongation factor G actively pushes both the decoding site and the mRNA/tRNA complex during translocation.

RNA scaffolds for minihelix-based aminoacyl transfer: design of "transpeptizymes".

Abstract Some evidence and considerations suggest that RNA minihelices based on the acceptor-TΨC stem-loop of tRNAs are the historical, more ancient part of the tRNA structure. These minihelices are substrates for aminoacylation by tRNA synthetases. In the transition from the RNA world to the theatre of proteins, aminoacyl minihelices may have had a role in early systems of peptide synthesis. Such systems would require bringing together aminoacyl groups into close proximity in order for peptide bonds to form. Here we report the design of RNA scaffolds based on pieces of the structure of the P4-P6 domain of the Tetrahymena ribozyme. RNA minihelices were incorporated into these scaffolds and the resulting RNAs could be enzymatically aminoacylated. The RNA scaffolds containing the minihelix-like pieces associated spontaneously to create the presumptive P4-P6 structure and thereby bring together the substrates for aminoacylation. Thus, peptide synthesis with associating RNA scaffolds that contain minihelix-like motifs appears plausible.

Major groove binding of the tRNA/mRNA complex to the 16 S ribosomal RNA decoding site.

We propose a detailed three-dimensional model, with atomic detail, for the structure of the Escherichia coli 16 S rRNA decoding site in a complex with mRNA and the A and P-site tRNAs. Model building began with four primary assumptions: (1) A and P-site tRNA conformations are identical with those seen in the tRNA crystal structure; (2) A and P-site tRNAs adopt an S-type orientation upon binding mRNA in the ribosome; (3) A1492 and A1493 bind non-specifically to the mRNA through a series of hydrogen bonds; and (4) C1400 lies in close proximity to the P-site tRNA wobble base in order to satisfy a UV-induced photocrosslink formed between the two residues. We have models with both major groove and minor groove binding of the tRNA/mRNA complex to the decoding site RNA, and conclude that major groove binding is more likely. Both classes of models maintain structural features reported in the NMR structure of the A-site region of the decoding site RNA with bound paromomycin. We also present models for the tRNA/mRNA complex bound to the decoding site RNA in the presence of the aminoglycoside paromomycin. We discuss possible mechanisms for ribosomal proof reading and antibiotic disruption of this proofreading.

Exploring three-dimensional structures of the HIV-1 RNA/tRNALys3 initiation complex.

Human immunodeficiency virus type 1 (HIV-1) uses host tRNA as a primer for reverse transcription of its viral RNA. The 3' terminal 18 nucleotides of human tRNALys3 are complementary to the primer binding site on the viral RNA. A secondary structure model for the HIV-1 RNA/tRNALys3 initiation complex has been proposed that includes additional base-pairing between the tRNA and the HIV-1 RNA beyond the 18 nucleotides of the primer binding site. Included in these interactions is base-pairing between the anticodon of tRNALys3 and an A-rich loop in the HIV-1 secondary structure. The tRNA and HIV-1 RNA are significantly unfolded from their native structures in order to form the initiation complex proposed in this model. We have found several problems with the proposed secondary structure in our efforts to build a three-dimensional model that is compatible with it. The additional interactions between the tRNA and viral RNA cause the structure to be topologically knotted. This poses a problem for folding of the initiation complex and transcription by reverse transcriptase. We have also not been able to build any all-atom models based on known RNA structures that follow the secondary structure model in the extended tRNA/HIV-1 RNA complex. Finally, beyond the primer binding site interaction, subsequent biochemical and genetic studies have given further insight into the structure of the initiation complex. These results call into question some of the extended HIV-1 RNA/tRNA interactions that have been proposed.

To knot or not to knot? Examination of 16S ribosomal RNA models.

The presence of topological knots in large RNA structures is highly unlikely given that 1) no RNA structures determined thus far contain topological knots, 2) secondary structure maps for most RNA molecules are knot free, 3) there are no known RNA topoisomerases, and 4) it is difficult to imagine how knots could be formed specifically and uniquely during transcription. Since native RNA structures probably lack topological knots, models of these RNA molecules should be free of knots as well. Therefore, we have examined four existing models for the 30S ribosomal subunit to determine if any of the three domains of the 16S rRNA molecule is knotted. We found that all but one model had at least one knotted domain. We conclude that models of large RNA molecules should be examined for knotting before publication.