scholarly journals Site-Directed Mutagenesis Studies of Tn5 Transposase Residues Involved in Synaptic Complex Formation

2007 ◽  
Vol 189 (20) ◽  
pp. 7436-7441 ◽  
Author(s):  
Soheila Vaezeslami ◽  
Rachel Sterling ◽  
William S. Reznikoff
Keyword(s):  
Complex Formation ◽  
Catalytic Reactions ◽  
Site Directed Mutagenesis ◽  
Dna Interactions ◽  
Synaptic Complex ◽  
Biochemical Assays ◽  
Protein Dna Interactions ◽  
End Sequences ◽  
Tn5 Transposase ◽  
Cocrystal Structure

ABSTRACT Transposition (the movement of discrete segments of DNA, resulting in rearrangement of genomic DNA) initiates when transposase forms a dimeric DNA-protein synaptic complex with transposon DNA end sequences. The synaptic complex is a prerequisite for catalytic reactions that occur during the transposition process. The transposase-DNA interactions involved in the synaptic complex have been of great interest. Here we undertook a study to verify the protein-DNA interactions that lead to synapsis in the Tn5 system. Specifically, we studied (i) Arg342, Glu344, and Asn348 and (ii) Ser438, Lys439, and Ser445, which, based on the previously published cocrystal structure of Tn5 transposase bound to a precleaved transposon end sequence, make cis and trans contacts with transposon end sequence DNA, respectively. By using genetic and biochemical assays, we showed that in all cases except one, each of these residues plays an important role in synaptic complex formation, as predicted by the cocrystal structure.

scholarly journals Protein-DNA Interactions at the Opossum Npt2a Promoter are Dependent upon NHERF-1

2016 ◽  
Vol 39 (1) ◽  
pp. 1-12 ◽  
Author(s):  
Barbara J. Clark ◽  
Rebecca D. Murray ◽  
Sarah A. Salyer ◽  
Samuel C. Tyagi ◽  
Cibi Arumugam ◽  
...  
Keyword(s):  
Complex Formation ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Proximal Promoter ◽  
Mrna Levels ◽  
Dna Interactions ◽  
Phosphate Homeostasis ◽  
Protein Levels ◽  
Protein Dna Interactions ◽  
Mrna Half Life

Background/Aims: Phosphate homeostasis is controlled by the renal reabsorption of Pi by the type IIa sodium phosphate cotransporter, Npt2a, which is localized in the proximal tubule brush border membrane. Regulation of Npt2a expression is a key control point to maintain phosphate homeostasis with most studies focused on regulating protein levels in the brush border membrane. Molecular mechanisms that control Npt2a mRNA, however, remain to be defined. We have reported that Npt2a mRNA and protein levels correlate directly with the expression of the Na+/H+ exchanger regulatory factor 1 (NHERF-1) using opossum kidney (OK) cells and the NHERF-1-deficient OK-H cells. The goal of this study was to determine whether NHERF-1 contributes to transcriptional and/or post-transcriptional mechanisms controlling Npt2a mRNA levels. Methods: Npt2a mRNA half-life was compared between OK and NHERF-1 deficient OK-H cell lines. oNpt2a promoter-reporter gene assays and electrophoretic mobility shift assays (EMSA) were used identify a NHERF-1 responsive region within the oNpt2a proximal promoter. Results: Npt2a mRNA half-life is the same in OK and OK-H cells. The NHERF-1 responsive region lies within the proximal promoter in a region that contains a highly conserved CAATT box and G-rich element. Specific protein-DNA complex formation with the CAATT element is altered by the absence of NHERF-1 (OK v OK-H EMSA) although NHERF-1 does not directly contribute to complex formation. Conclusion: NHERF-1 helps maintain steady-state Npt2a mRNA levels in OK cells through indirect mechanisms that help promote protein-DNA interactions at the Npt2a proximal promoter.

Sign in / Sign up

Export Citation Format

Share Document