PubMed · 4288166
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L J DeGroot, A D Dunn, S Jaksina. 1966. Thyroid mitochondrial respiration.. https://doi.org/10.1210/endo-79-1-28
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UAP56 (U2AF65 associated protein) is a member of the DEAD-box helicase family. Helicases are essential enzymes generally involved in the metabolism of nucleic acids. The gene encoding a member of DEAD-box family was cloned and characterized from the human malaria parasite Plasmodium falciparum. PfU52 is homologous to UAP56 and contains the RNA-dependent ATPase, RNA helicase and RNA binding activities. Using the parasite extract we report that PfU52 is involved in splicing reaction. Site-directed mutagenesis studies indicate that the conserved residues glycine 181, isoleucine 182 and arginine 206 are involved in RNA binding and this activity is required for the enzymatic activities of PfU52. PfU52 is expressed in all the intraerythrocytic developmental stages of the parasite. In the present study we have reported the detailed characterization of PfU52 from P. falciparum and these results advance the knowledge regarding the function of UAP56 in general.
Ydj1 is the major type I Hsp40 (heat-shock protein 40) family member in yeast. Ydj1 can pair with yeast Hsp70 Ssa1 to facilitate protein translocation and protein folding. Ydj1 itself can also function as a molecular chaperone to bind the non-native polypeptides and suppress protein aggregations in vitro. The crystal structure of Ydj1 complexed with its peptide substrate GWLYEIS reveals that a hydrophobic pocket located on Ydj1 domain I may play a major role in mediating the interactions between Ydj1 and the peptide substrate. To understand the mechanism by which Ydj1 interacts with non-native polypeptide, we have mutated the residues forming the hydrophobic pocket, based on the structural information. We have also constructed deletion mutations of the zinc-finger motifs within Ydj1. We have examined the functional consequences of these Ydj1 mutants by in vivo and in vitro assays. The results indicated that the hydrophobic pocket located on Ydj1 plays a critical role in its molecular chaperone activity by mediating interactions with the non-native polypeptides.
Haemophilus influenzae DNA mismatch repair proteins, MutS, MutL and MutH, are functionally characterized in this study. Introduction of mutS, mutL and mutH genes of H. influenzae resulted in complementation of the mismatch repair activity of the respective mutant strains of Escherichia coli to varying levels. DNA binding studies using H. influenzae MutH have shown that the protein is capable of binding to any DNA sequence non-specifically in a co-operative and metal independent manner. Presence of MutL and ATP in the binding reaction resulted in the formation of a more specific complex, which indicates that MutH is conferred specificity for binding hemi-methylated DNA through structural alterations mediated by its interaction with MutL. To study the role of conserved amino acids Ile213 and Leu214 in the helix at the C-terminus of MutH, they were mutated to alanine. The mutant proteins showed considerably reduced DNA binding and nicking, as well as MutL-mediated activation. MutH failed to nick HU bound DNA whereas MboI and Sau3AI, which have the same recognition sequence as MutH, efficiently cleaved the substrate. MutS ATPase activity was found to be reduced two-fold in presence of covalently closed circular duplex containing a mismatched base pair whereas, the activity was regained upon linearization of the circular duplex. This observation possibly suggests that the MutS clamps are trapped in the closed DNA heteroduplex. These studies, therefore, serve as the basis for a detailed investigation of the structure-function relationship among the protein partners of the mismatch repair pathway of H. influenzae.