mRNA interferases are sequence-specific endoribonucleases encoded by toxin-antitoxin (TA) systems in bacterial genomes. Previously, we demonstrated that Mycobacterium tuberculosis contains at least seven genes encoding MazF homologues (MazF-mt1 to -mt7) and determined cleavage specificities for MazF-mt1 and MazF-mt6. Here we have developed a new general method for the determination of recognition sequences longer than three bases for mRNA interferases with the use of phage MS2 RNA as a substrate and CspA, an RNA chaperone, which prevents the formation of secondary structures in the RNA substrate. Using this method, we determined that MazF-mt3 cleaves RNA at UU CCU or CU CCU and MazF-mt7 at U CGCU ( indicates the cleavage site). As pentad sequence recognition is more specific than those of previously characterized mRNA interferases, bioinformatics analysis was carried out to identify M. tuberculosis mRNAs that may be resistant to MazF-mt3 and MazF-mt7 cleavage. The pentad sequence was found to be significantly underrepresented in several genes, including members of the PE and PPE families, large families of proteins that play a role in tuberculosis immunity and pathogenesis. These data suggest that MazF-mt3 and MazF-mt7 or other mRNA interferases that target longer RNA sequences may alter protein expression through differential mRNA degradation, a regulatory mechanism that may allow adaptation to environmental conditions, including those encountered by pathogens such as M. tuberculosis during infection.