Lysine 100 acetylation effect of CRP (catabolite activator protein) in gene expression in Escherichia coli. Escherichia coli K-12

cAMP receptor protein (CRP, also known as the catabolite activator protein [CAP]) is arguably the best-studied of the global transcription factors of E coli. CRP alone is responsible for regulating at least 283 operons. Upon binding cAMP, the CRP dimer binds DNA and directly interacts with RNA polymerase (RNAP). At Class II promoters, CRP binds near position -41,5 relative to the transcription start site and contacts the amino-terminal domain of the RNAP α subunit (RNAPα-NTD). This interaction requires AR2, a patch of primarily positively charged residues (H19, H21, E96, and K101) that interact with negatively charged residues on RNAPα-NTD. Acetylome analyses consistently detect lysine 100 (K100) of CRP as acetylated. Since K100 is adjacent to the positively charged AR2, we hypothesized that the K100 positive charge may also play a role in CRP function. We further hypothesized that acetylation of K100 would neutralize this positive charge, leading to a potential regulatory mechanism Overall design: To determine the effect of the K100 positive charge of CRP on promotes Class II transcription, we inset the K100R (named R) and K100Q (named Q) mutations into the chromosome. Since we were unable to insert these mutations into the native crp locus, we placed crp alleles (along with the native promoter region) into the paaH gene locus in a Δcrp background. Therefore, the insertion of native crp gene was also conducted into same position, and it was used as control, named as N. To this end, we performed RNA microarray on strains WT N, Q, or R variants of CRP grown in M9 minimal medium with 10mM glucose or 30mM acetate as sole carbon source and harvested during exponential growth or after entry into stationary phase. We used two biological replicates in this study and each condition (named 1 and 2).