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The non-coding small RNA, IhtA expressed by the obligate intracellular human pathogen  Chlamydia trachomatis  modulates the translation of HctA, a key protein involved in replicative to infectious cell type differentiation. Using a combination of bioinformatics and mutagenesis we sought to identify the base pairing requirement for functional repression of HctA protein expression, with an eye to applying our findings towards the identification of additional targets. IhtA is predicted to fold into a three stem:loop structure. We found that loop 1 occludes the initiation codon of  hctA , while loop 2 and 3 are not required for function. This 7 nucleotide region forms G/C rich interactions surrounding the AUG of  hctA . Two additional genes in the chlamydial genome,  CTL0322  and  CTL0097 , contained some elements of the  hctA :IhtA recognition sequence. The mRNA of both  CTL0322 and  CTL0097  interacted with IhtA in vitro as measured by biolayer interferometry. However, using a CheZ reporter expression system, IhtA only inhibited the translation of  CTL0322 . The proposed IhtA recognition site in the  CTL0322  message contains significant G/C base pairing on either side of the initiation codon while  CTL0097  only contains G/C base pairing 3’ to the AUG initiation codon. These data suggest that as the functional interacting region is only 6-7nt in length that full translation repression is dependent on the degree of G/C base pairing. Additionally our results indicate that IhtA may regulate multiple mRNAs involved in the chlamydial infectious cycle.

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Identification of the Base-Pairing Requirements for Repression of hctA Translation by the Small RNA IhtA Leads to the Discovery of a New mRNA Target in Chlamydia trachomatis