Link to this post | posted 02 May, 2023 03:58

fbaliraine

In my previous post entitled “Is there any recent evidence of a tRNA overlapping a protein gene, even by a few bp?” (https://seaphages.org/forums/topic/5365/) I thought this question was settled. I want to delete the gene in phage Glaske16 at position 60940-61320 bp, but because several recent annotations have kept it, and it has more than 70 hits to the HNH endonuclease in phagesDB, I am seeking a second opinion on this. Its sequence is MQREYMRRWVANRRSAFFASKQCAMCGAGEELELDHIDPTKKVDHRIWSWTDARRSEELAKCQVLCASCHKKKTGEQWYANRSVSENAHHGTSRRYRKMKCRCGLCRLGNTNRSRALRQRHRVPVE Despite more than 70 hits to HNH endonuclease in phagesDB, this gene has low (<50%) coding potential in Genemark_S, and entirely no CP in Genemark_smeg, and TB. THIS GENE OVERLAPS 15bp WITH A tRNA CALLED BY ARAGORN v1.2.41 AND tRNA-SE v. 2.0. WITH AN INFERNAL SCORE OF 55.5. I am asking this question because of the statement from the Resource Guide entitled, “Predicting tRNA and tmRNA genes” (https://seaphagesbioinformatics.helpdocsonline.com/article-40): “It is highly unusual that a phage tRNA willi) Be encoded within an ORF called by Glimmer and GeneMark that has high coding potential, (ii) Be encoded on the opposite strand as a number of other phage tRNAs found in the same genome, (iii) Be encoded at a genomically distant location from the other tRNA genes in a genome. In general, violation of any of the three preceding conditions is sufficient for exclusion of a potential tRNA from an annotation (we have found a single high scoring tRNA that is not part of the rest of the large cluster, however this situation is very rare).” Whereas I am inclined to delete the protein coding gene and keep the tRNA since it is called by ARAGORN and tRNAscan-SE with a high infernal score (55.5), I also note that this tRNA is distant from other tRNAs, being 387 bp apart, which seems to violate caveat iii above. According to the forum post, “How close can one pack protein and tRNA's genes” of Feb 24, 2016, Dr Pope stated that, “We tend to steer clear of a tRNA and a protein occupying the same space, but there are definitely genomes where they get pretty close.” I realize that there may be exceptions though but wanted to be sure. Some M1 phages such as Reindeer & Iphrane7 do not have this gene but have the “Glu” tRNA (61306-61380 bp in Glaske16; see figures & DNA Master file attached), and we know that tRNAs tend to be conserved. Edited 02 May, 2023 04:16 346Kb 67Kb

Link to this post | posted 21 Jul, 2022 18:29
fbaliraine	Fabulous! Thank you Karen! Fred

Link to this post | posted 15 Jul, 2022 21:44

fbaliraine

Phage Skinny gene at 63560-63949 bp hits several LAGLIDADG endonuclease, homing endonuclease, HNH endonuclease, with low similarity (less than 60%). Wondering whether to call it with the general “Hydrolase” or “NKF”? The amino acid sequence is: MDLAYLGGFFDGEGNVGLYKSGGESPRLRVQVFQNHGASQDRLMHEIHDTFGGTLHDRGTGYLYSASGSRAVDLLTQLRPHLRLKLEQADEALEWWRNRTAERFRSRTAEEVAYDESAMTRLKELKRAGZ I have also attached the relevant BLASTp and HHPred data: Thanks! 460Kb

Link to this post | posted 09 May, 2022 19:05

fbaliraine

Thanks. I’ve looked at the “Thioredoxin” link that you’ve kindly provided, but there is something noteworthy in HHPred. In PDB, the reference sequence phage Onyinye gene78 for “oxidoreductase” is almost thrice as long 792 bp vs 273 bp of Gilberta) and mostly hits “Polyketide oxygenase PgaE,” chain A, with ref Sequence in pfam hitting the "FAD folding domain" but I do not see any hits in Gilberta to any "FAD folding domain." Instead, in PDB Gilberta hits the "Molecule" THIOREDOXIN chains A & B, with the pfam ref hitting “Glutaredoxin or Glutaredoxin-like NRDH-redoxin, THIOREDOXIN; OXIDOREDUCTASE, GLUTAREDOXIN.” On the other hand, the ref sequence for Thioredoxin, phage Cjw1 gp 37 (246 bp) has a comparable length to Gilberta’s 273 bp and they both have several hits the molecule Thioredoxin chain A, but not the “Polyketide oxygenase PgaE,” or “FAD domain”. Edited 09 May, 2022 19:08

Link to this post | posted 08 May, 2022 02:08

fbaliraine

There is a "mixed bag" with many hits to NrdH-like glutaredoxin and glutaredoxin in phagesDB, with a few hits to Thioredoxin for this Gilberta sequence MRTMFAPITIYTQPRCAPCDALKKRLEKEGIAFDAVDITKNEEAYAYVTGVLKAAATPIIVTDTHDPIIGDRPAELEELIEYYTTSETGVZ However, the PDB HHPred shows more than 22 hits to Thioredoxin with alignments of 78-93% and probability of 99%, versus less than 5 hits to NrdH-like glutaredoxin with alignments ranging from 81-85% and probability of 99%, besides hits to glutaredoxin, all three of which are options currently provided in the Official Functions list. Should we go for, “Thioredoxin” or use “NrdH-like glutaredoxin” or the general term "glutaredoxin"? In the “Cluster EB/ED glutaredoxin” forum post of 06 May, 2019, the use of “NrdH-like glutaredoxin” was considered appropriate for “HHPRED hit with >98% prob and >98% coverage to 4FIW_A which is the published crystal structure of NrdH from E coli.” See details of hits below: HHPred PDB hits to Thioredoxin (6MOS_A, 93.4% alignment, 99.14% probability; 7ASW_A, 91.2% alignment, 98.91% probability; 3ZIT_B, 84.62% alignment, 99.47% probability; d1nhoa_, 85.71 alighment, 99.23% probability; d1f9ma_, d1ti3a_, d1r26a1, & d1ep7a_, 86.81 alignment, 99.2% probability; d4oo4a_, 85.71 alignment, 99.18% probability; d4j56e1 & d1thxa_, 85.71 % alignment, 99.13% probability; 3KP8_A, 84.61% alignment, 99.12% probability; d1nw2a_, 84.61% alignment, 99.02% probability; d1iloa_ c, 78.02% alignment, 99.1% probability; d3diea1, 83.52% alignment, 99.1% probability; 7B02_A, 86.81% alignment, 99.1% probability; 3HZ4_A & 7RGV_A, 90.1% alignment, 99.1% probability; 7BZK_B, 6ZYW_P, 6I1C_B, & 6Q6T_A, 89.01% alignment, 99.0% probability; 1THX_A, 87.91% alignment, 99.02% probability). HHPred hits to NrdH-like glutaredoxin in (d1r7ha_, 81.32% alignment, 99.49% probability; d1h75a_, 83.52% alignment, 99.46 probability; 1R7H_A, 81.32% alignment, 99.11% probability; 4K8M_A, 84.61% alignment, 98.99% probability). Thanks! Edited 08 May, 2022 02:16

Link to this post | posted 06 May, 2022 18:27
fbaliraine	Thank you Debbie! Fred

Link to this post | posted 06 May, 2022 17:34

fbaliraine

In the subcluster A11 phage Gilberta, we are seeing a small (189 bp long; position 25381-25569) gene hitting more than 60 minor tail protein genes in both NCBI and phagesDB. This gene is right downstream of a large (1992 bp long) minor tail protein gene which follows other minor tail proteins upstream of it. However, this small (189 bp) gene has neither hits to collagen-like, glycine-rich proteins, coiled-coils, nor any other significant hits in HHPred (Only one HHPred hit to Bacteriophage FRD2 protein, with 41.2% alignment and 11.9% probability). Could we still call it a minor tail protein solely based on synteny? Below is its amino acid sequence: MPWSPSPAFPQRQHRTAWFAELPAPTPAQHQTAWWAVYELDAPVEIACVTAAEGQEGPEEAVZ Thanks! Fred Edited 06 May, 2022 19:16

Link to this post | posted 21 Apr, 2022 23:19
fbaliraine	Hi Debbie, I concur. Case closed! Thank you for critically looking at this and clearing the air about calling genes that overlap with tRNAs. Perhaps a note in the resource guide can help eliminate future possibilities of someone calling such protein genes simply based on previous calls or significant BLASTp matches. Fred

Link to this post | posted 19 Apr, 2022 02:30

fbaliraine

Hi Debbie, Please find the DNA Master file attached. The tRNAs look legit, but the question is whether to delete the reverse gene at 92938-93090 bp, given that it is currently in pham 14023 with 31 members and hits various phage genes (including very recently annotated phages such as Ronan gp 160, GenBank submission 01-JUN-2020, and Mangeria GenBank Accn # YP_010057792.1) in phagesDb with q1:s1, 100% identity and e-values such as 6e-22. Thanks, Fred 811Kb

Link to this post | posted 14 Apr, 2022 21:52

fbaliraine

I am asking this question to have this issue settled and possibly suggest that a note be included in the Resource Guide regarding tRNA annotation. Phage IkeLoa reverse draft gene 134 at 92938-9390 bp overlaps a tRNA by 27 bp; even if we change the start, it still overlaps by a few bp. Its sequence is: MLYQLSYDGGAAGETSQPSRLRVMSPVCLPSTTIPARVTDEDLTFVKPLSZ According to the forum post, “How close can one pack protein and tRNA's genes” of Feb 24, 2016, Dr Pope stated that, “We tend to steer clear of a tRNA and a protein occupying the same space, but there are definitely genomes where they get pretty close.” We have been following this rule and I am inclined to delete this protein gene, but I want to be sure in case something has changed recently, because it is matching q1:s1, 100% with recently annotated genes in phagesDB and phamerator (years 2017-2020; see attached). Thanks! Fred 267Kb