1. Evaluation Summary:

    This manuscript provides the first 3D structure of a novel type of RNA processing enzyme recently identified in bacteria. It convincingly uses cryoEM and biochemistry to describe how this small enzyme makes a new type of homo polymeric complex as required for its activity. The manuscript provides important conceptual novelties that will be of interest for a broad readership of biologists interested in gene expression processes.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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  2. Joint Public Review:

    Nature has evolved remarkably different enzymes for the essential processing of 5´ ends of pre-tRNAs. The ribonucleoprotein RNase P uses its RNA component for pre-tRNA recognition and catalysis, the protein-only RNase P (PRORP) contains a pentatricopeptide repeat domain for pre-tRNA recognition and a nuclease domain for catalysis, and more recently a new family, Homolog of Aquifex RNase P (HARP), was identified. The HARPs seem mysterious as they are quite small (~23 kDa) and form oligomers. Although they appeared to possess a catalytic domain, it was unclear how they would recognize and process pre-tRNAs. Here the authors have addressed these questions by determining a cryo-EM structure of a dodecameric HARP, Hhal2243, and using the structural information to strikingly demonstrate the essential nature of the oligomerization for enzymatic activity of the Aquifex HARP, Aq880. Enzymatic activity assays with mutant enzymes identify basic residues for pre-tRNA substrate recognition, and a preliminary HARP/tRNA model suggests a possible mode for pre-tRNA recognition and catalysis by the dodecamer.

    The cryo-EM model illustrates the overall formation of a dodecamer structure with six dimers rotated about a central screw axis. The structure of Hhal2243 was used to design C-terminal deletion mutants of Aq880 that would disrupt inter-dimer interactions. The authors used mass photometry to identify the distribution of oligomer sizes for wild-type and C-terminally truncated Aq880 and measured the enzymatic activities of the wild-type and truncated Aq880. These combined data convincingly demonstrated that enzymatic activity is lost when truncation eliminates the dodecamer form. A compelling strength of the manuscript is the correspondence of the enzymatic activity and the rich information on oligomerization from the mass photometry.

    The authors aligned their cryo-EM model with the crystal structure of the Arabidopsis PRORP1 to show that the arrangement of catalytic acidic residues is conserved in these two families, although we believe the overall structure of each protein family is distinct. To clarify this point, please explain the structural alignment more (page 9, lines 178-179). Are the folds distinct, yet the catalytic residues align? The authors should consider moving Fig. S5 to the main figures. The conservation of the arrangement of catalytic residues will likely be of interest to enzymologists fascinated by the consistent geometric arrangement produced by distinct structural scaffolds.

    With the identification of the processing active site and R125 and R129 as a substrate recognition site, the authors attempted to model pre-tRNA engagement by the dodecamer. The model is rather speculative at this stage, but the authors placed this analysis in the Discussion, which seems appropriate, and it develops a testable model for future work. We did, however, find it somewhat confusing to understand which monomers were engaging pre-tRNA, and we recommend improving the presentation of the model and how it was generated.

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