Remote homology identification of the Drosophila melanogaster ortholog of the RNA Polymerase I subunit Rpa34/POLR1G

Abstract

Highly conserved orthologous proteins are easily identified by sequence homology alone, whereas poorly conserved orthologs require additional structural information to be identified. All Drosophila orthologs of RNA polymerase I, II, and III subunits-except one-have been identified by sequence homology. Here, we identified CG11076 as the missing Rpa34/POLR1G ortholog in Drosophila . Remote homology detection and secondary structure analysis showed that CG11076 is predicted to have high structural conservation with Rpa34/POLR1G, and phylogenetic analysis demonstrated that these proteins are closely related. Our work underscores the importance of utilizing both sequence and structure to identify highly divergent orthologous proteins in different species.

Figure 1. CG11076 is the Drosophila ortholog of yeast Rpa34 and human POLR1G

(A-B) HHpred results for S. cerevisiae Rpa34 (ScRpa34) against the Drosophila proteome (A) and Drosophila CG11076-PA (DmCG11076-PA) against the PDB database (B). Topmost bar represents the domain structure of the query protein. CTD: carboxy-terminal domain. The bottom three bars represent the target proteins. Target bars overlap the region of the query that was aligned by HHpred. P: probability (the likelihood of a positive match between the query and target), E: E-value (the average number of nonhomologous hits expected), I: percent identity (percent of identical residues between the query and target), F: percent fold (the percent of equivalent query and target residues predicted to have a similar secondary structure). (C) Multi Sequence Alignment (MSA) of ScRpa34, human POLR1G (HsPOLR1G), and DmCG11076-PA using HHpred. Residues highlighted in blue are identical in at least two sequences. Residues that are identical in all three sequences are marked by an asterisk. Note that the long, disordered C-terminal domain of HsPOLR1G was not included in the MSA to prevent erroneous gaps in the MSA. (D) FELLS secondary structure analysis and disorder prediction of DmCG11076-PA. (E-F) DmCG11076-PA modeled onto the Cryo-EM structures of ScRpa34 (E) and HsPOLR1G (F) using MODELLER. Alignment and RMSD calculations were made in PyMol. (G) AlphaFold predicted structure of DmCG11076-PA, colored by confidence (b-factor) in PyMol. (H) Alignment of the beta sheet segments of the AlphaFold predicted structures of ScRpa34, DmCG11076-PA, and HsPOLR1G. Alignment and RMSD calculations were made in PyMol. (I) Phylogenetic analysis of Rpa34 orthologs and paralogs from S. cerevisiae , Drosophila , and human.