Fig 4. Identification of key amino acid residues of TRAM0076 required for nucleic-acid unfolding activity. ( A ) Structural comparison between E . coli CspA (left) and TRAM0076 (right) predicts the potential substrate binding surface and the key residues (sticks) in TRAM0076 equivalent to the residues essential for CspA unfolding activity. These residues are indicated by stars underneath the sequences in Fig B . Red sticks indicate the key residues for TRAM0076’s structured RNA unfolding activity in Fig C and the growth complement in Fig D . ( B ) Sequence alignment of TRAM proteins from some Archaea. Amino acids that are identical (red shadowed), highly conserved (framed) and conserved (orange letters) are indicated. Arrows and 0 TT 0 indicate the β -sheets and turns, respectively. Asterisks indicate the amino acids selected for substitutions. ( C ) Transcriptional antitermination assay of the TRAM0076 mutants in E . coli RL211. Using the same procedure as in Fig 3 B , growth of E . coli RL211 cloned with each of the MMP0076 mutants was determined on LB plates plus (+Cm) or minus (-Cm) chloramphenicol. ( D ) Complementation in M . maripaludis by the substitution mutants in the MMP0076 deletion strain ( Δ 0076 ). Growth was measured at 37˚C for triplicate cultures. Averages and standard deviations are