No description available.

Conformational stability of the collagen triple helix affects its turnover and determines tissue homeostasis. Although it is known that the presence of imino acids (prolines or hydroxyprolines) confer stability to the molecule, little is known regarding the stability of the imino-poor region lacking imino acids, which plays a key role in collagen cleavage. In particular, there have been continuing debates about the role of water in collagen stability. We addressed these issues using molecular dynamics simulations on 30-residue long collagen triple helices, including a structure that has a biologically relevant 9-residue imino-poor region from type III collagen (Protein Data Bank ID: 1BKV). We characterized the conformational motion of the molecule that differs between imino-rich and imino-poor regions using a torsional map approach. At temperatures of 300 K and above, unwinding initiates at a common cleavage site, the glycine-isoleucine bond in the imino-poor region. This provides a linkage between previous observations that unwinding of the imino-poor region is a requirement for collagenase cleavage, and that isolated collagen molecules are unstable at body temperature. Unwinding of the imino-poor region is controlled by dynamic water bridges between backbone atoms with average lifetimes on the order of a few picoseconds, as the degree of unwinding strongly correlated with the loss of water bridges, and unwinding could be either prevented or enhanced, respectively by enforcing or forbidding water bridge formation. While individual water bridges were short-lived in the imino-poor region, the hydration shell surrounding the entire molecule was stable even at 330 K. The diameter of the hydrated collagen including the first hydration shell was about 14 Â, in good agreement with the experimentally measured inter-collagen distances. These results elucidate the general role of water in collagen turnover: water not only affects collagen cleavage by controlling its torsional motion, but it also forms a larger-scale lubrication layer mediating collagen self-assembly.

There are numerous RNAs transcribed in the cell that are not directly involved in protein translation. Maintaining proper levels of RNA is crucial for cell viability, making RNA surveillance an essential process (equivalent to regulating protein levels). Mtr4 is an essential RNA helicase that activates exosome-mediated 3'-5' turnover in RNA processing mechanisms. Mtr4 has several binding partners, with the most prominent one being the complex Trf4/5-Air1/2-Mtr4 polyadenylating (TRAMP) complex. The polyadenylation and unwinding activity of TRAMP is modulated by a sensing mechanism in Mtr4 that detects both length and identity of 3'-end poly(A) tails. While it has been known that Mtr4 has an unwinding preference for substrates with a 3' poly(A) tail and a length of approximately 5 nucleotides, the mechanistic detail is unclear. It is also unclear what structural features of Mtr4 contribute to this sensing function. By using x-ray crystal structures of Mtr4, a ratchet helix was identified to interact with RNA substrates. Significant conservation of this ratchet helix along the RNA binding path was observed, similar to conservation patterns throughout Ski2-like and DEAH/RHA-box helicases. Structural characterization revealed a novel arch domain shown to bind structured RNAs, which may aid in cooperative RNA recognition in conjunction with the ratchet helix. In this thesis we demonstrate that the conserved residues at the third (R1030) and fourth (E1033) turns of the Mtr4 ratchet helix uniquely influence RNA unwinding rates. Furthermore, when mutated, ratchet helix positions confer slow growth phenotypes to Saccharomyces cerevisiae and are synthetically lethal in an Mtr4-archless background. The unwinding activity of these mutants when in the TRAMP complex alters the unwinding rates of Mtr4, and in some instances recovers substrate specificity. Our findings demonstrate the importance of R1030 and E1033 for helicase activity, and additionally link the arch domain of Mtr4 in essential unwinding events.

No / The design and synthesis of a potentially more therapeutically-viable azinomycin analogue 4 based upon 3 has been completed. It involved coupling of a piperidine mustard to the acid chloride of the azinomycin chromophore. Both the designed azinomycin analogue 4 and the natural product 3 bind to DNA and cause unwinding, supporting an intercalative mode of binding. Graphical abstract A designed analogue of the left half of azinomycin has been synthesized and unwinds supercoiled DNA.

Ribonucleases play essential roles in RNA metabolism. In Escherichia coli, the extensive degradation of RNAs that are defective or no longer required by the cell is carried out by one of three processive, 3' to 5' exoribonucleases. Relatively unstructured mRNAs are typically degraded by RNase II or PNPase. In contrast, mRNAs containing extensive secondary structure, and the highly structured rRNA and tRNA molecules, are degraded by PNPase and/or RNase R. However, RNase R differs from other exoribonucleases in that it is able to degrade through these structured RNAs without the aid of a helicase activity. Consequently, its mechanism of action is of great interest. In this dissertation, using a variety of specifically designed substrates, I show that a single-stranded overhang, which must be at least 5 nucleotides in length, is required for tight binding and subsequent degradation of double-stranded RNA by RNase R. Moreover, this overhang must be 3' to the duplex indicating that an RNA substrate must thread into the enzyme with 3' to 5' polarity. Using a series of truncated RNase R proteins, I show that the cold-shock domains and the S1 domain contribute to substrate binding. The cold-shock domains appear to play a role in substrate recruitment, while the S1 domain is required to position substrates for efficient catalysis. Furthermore, the nuclease domain alone is sufficient to bind and degrade structured RNAs. This is a unique property of the nuclease domain of RNase R since this domain in RNase II, a paralogue of RNase R, stalls as it approaches a duplex. RNase R binds RNA more tightly within its nuclease domain than RNase II. Through mutagenesis studies, I identify one amino acid, R572, within the nuclease domain of RNase R that contributes to this tight binding and the ability to degrade double-stranded RNA. Furthermore, I found that degradation of structured RNA is strongly dependent on temperature. Based on these data I propose that tight binding allows RNase R to capitalize on the natural thermal breathing of an RNA duplex to degrade structured RNA.

No description available.

No description available.

No description available.

No description available.

No description available.