Genetic Analysis of the Nitrogen Assimilation Control Protein from Klebsiella pneumoniae

Abstract

The nitrogen assimilation control protein (NAC) from Klebsiella pneumoniae is a typical LysR-type transcriptional regulator (LTTR) in many ways. However, the lack of a physiologically relevant coeffector for NAC and the fact that NAC can carry out many of its functions as a dimer make NAC unusual among the LTTRs. In the absence of a crystal structure for NAC, we analyzed the effects of amino acid substitutions with a variety of phenotypes in an attempt to identify functionally important features of NAC. A substitution that changed the glutamine at amino acid 29 to alanine (Q29A) resulted in a NAC that was seriously defective in binding to DNA. The H26D substitution resulted in a NAC that could bind and repress transcription but not activate transcription. The I71A substitution resulted in a NAC polypeptide that remained monomeric. NAC tetramers can bind to both long and shorter binding sites (like other LTTRs). However, the absence of a coeffector to induce the conformational change needed for the switch from the former to the latter raised a question. Are there two conformations of NAC, analogous to the other LTTRs? The G217R substitution resulted in a NAC that could bind to the longer sites but had difficulty in binding to the shorter sites, and the I222R and A230R substitutions resulted in a NAC that could bind to the shorter sites but had difficulty in binding properly to the longer sites. Thus, there appear to be two conformations of NAC that can freely interconvert in the absence of a coeffector.

A NAC for Regulating Metabolism: the Nitrogen Assimilation Control Protein (NAC) from Klebsiella pneumoniae

“…A comparable PC mutant of NAC, NAC H26D , has been isolated and shown to have a PC phenotype (67), so there is reason to suspect that many of the features of activation by LTTRs may be generalizable. However, it seems likely that H26 of NAC plays a more complex role in activation (15,67). NAC H26A is wild type for activation of hutUp, and several other drastic substitution mutants (e.g., NAC H26K and NAC H26Y ) also retain a substantial ability to activate hutUp (67).…”

“…NAC H26A is wild type for activation of hutUp, and several other drastic substitution mutants (e.g., NAC H26K and NAC H26Y ) also retain a substantial ability to activate hutUp (67). NAC activates ureDp from a site centered at Ϫ47, overlapping the RNA polymerase binding site and on the opposite face of the DNA helix from the site at hutUp (33), and yet NAC H26D shows a positive-control phenotype at this promoter as well (67). It seems unlikely that H26 could make the same kinds of contact with RNA polymerase in both cases.…”

“…Since NAC recognizes both sites without any coeffector signal to change its conformation (66), there was a question of whether NAC did in fact have two different conformations. Using the structures of OxyR and CbnR as a guide, mutants of NAC were generated that should have been unable to assume one or the other predicted conformation (67). One such mutant readily bound cod but not nac; the other readily bound nac but not cod.…”

Properties of the NAC (Nitrogen Assimilation Control Protein)-Binding Site within the ureD Promoter of Klebsiella pneumoniae

“…An analogous PC mutant of NAC, NAC H26D , has been isolated. NAC H26D is able to bind DNA and repress transcription but cannot activate either hutUp or ureDp (23). NAC can activate hutUp from an NBS at Ϫ64 and ureDp from an NBS at Ϫ47, on the face of the DNA helix opposite RNA polymerase.…”

“…This was even more surprising given a positive-control mutant (NAC H26D ), which by analogy to other LTTRs (10, 30) was initially thought to define the interaction region of LTTRs with the ␣ subunit of RNA polymerase. However, NAC H26D fails to activate both hutUp and ureDp (23), and it seems unlikely that amino acid H26 makes the same contact with ␣ from such very different positions on the DNA. That led us to examine whether there was something unique to the NBS ureD or the ureDp that differed from their hut counterparts.…”