Rv2516c Resolved · high

Annotation: from legacy to revised

Curation note: 2026-07-30 (hand review, feedback from the dedicated deep-dive project mtbc/Rv2516c). The previous auto-generated summary surfaced only the ribosomal-S6 Foldseek hit, which seeded the dedicated project on a single weakly-supported RNA-binding lead; four independent layers of this same fiche point instead to DNA binding (see function). Verdict deliberately LEFT AT 'dark': converging weak leads orient the experimental plan, they do not assign a function. Operon context worth noting: Rv2516c overlaps Rv2517c by 4 bp, and Rv2517c is modestly up-regulated under moxifloxacin at 2x MIC, in an experiment where the SOS regulon showed NO significant change at that dose, and its authors call it a persistence gene rather than an SOS gene (Iacobino A, Piccaro G, Pardini M, Fattorini L, Giannoni F, 2021, Microorganisms 9(2):255, doi 10.3390/microorganisms9020255). An earlier version of this note read that paper's TITLE as if it described the gene, and the resulting 'DNA-damage-responsive regulator' hypothesis rested on that misreading (P18.17). It is further weakened by measurement: across GSE71200 (100 arrays, 9 antibiotics, 6 modes of action) the induction of Rv2516c and Rv2517c is NOT specific to DNA damage - recA separates the classes cleanly (percentile 97 under moxifloxacin, 5-35 elsewhere) whereas Rv2516c sits as high under kanamycin (93) and capreomycin (94) as under moxifloxacin (91), with r(recA, Rv2516c) = -0.04. Read the pair as responding to antibiotic stress in general, and the predicted HTH as an unassigned lead. Note also that Rv2517c is NON-essential (NE, VI +0.54, CI crossing zero) where Rv2516c is essential by CRISPRi (VI -9.91, `certain` flag set) — Tn-seq places the requirement on a sub-region only (ESD: 5 of its 14 TA sites essential, 9 not), so the contrast rests on the knockdown screen: an obligate heterodimer is therefore unlikely, and an AF-Multimer test of the pair needs a positive control to be interpretable. 2026-09-01 (P8.14, resync from the dedicated project mtbc/Rv2516c/, iteration 20, phase193_gene_atlas_resync_rv2516c.py). Verdict moved dark -> requalified, confidence low -> high: the sub-threshold Sigma70_r4/HTH lead this fiche carried since 2026-07-30 is now resolved, by domain-isolated HHpred (unit B, aa 88-147, Probability 99.28%, E=1.3e-10, >4500x stronger than the full-length search), to the AlpA/excisionase/recombination-directionality-factor family (PF05930/COG3311, Wen et al. 2022 NAR). The ribosomal-S6 Foldseek reading that originally seeded the dedicated project is REFUTED. This is a fold/family assignment, not a demonstrated activity: no excisionase or antitermination activity, no operator and no base-specific DNA contact are shown for Rv2516c itself; two independent Boltz-2 co-folding tests failed as a discriminant test of DNA-binding sequence specificity (method failure, not biological negative), and the question stays open pending an experimental assay (EMSA/footprinting), outside the scope of computation. Full dossier (three-unit architecture, PAE-justified domain independence, cross-genus conservation gradient, operon and M. leprae deletion evidence, DosR exclusion): mtbc/Rv2516c/etat_des_decouvertes.md.

In the literature (TB corpus sweep) named only in full text

No TB publication mentions this locus tag in its title or abstract (sweep of a ~330k-abstract PubMed TB corpus). A full-text search does however find 1 paper(s) naming this gene in their body text — see the full-text lead field below (the gene is listed there only as an output of a computational regulatory-network model, with no experimental validation specific to it). The gene is therefore not unmentioned, and this page no longer claims that it is unstudied.

Scope of the sweep: What was actually swept: the H37Rv locus tag and the ortholog locus tags (M. bovis Mb…, M. marinum MMAR_…, M. smegmatis MSMEG_…, M. leprae ML…, M. abscessus MAB_…), plus the gene name where one exists. NOT swept: protein and enzyme names, including family shorthands such as Ldt(Mt4) or VapC28, and their orthographic variants. An absent locus-tag mention is therefore evidence about the TAG, and only indirect evidence about the protein (P18.14).

An antigen status or a virulence phenotype is NOT a molecular function: the verdict stays unchanged. This layer adds the missing context, it does not requalify the gene. This distinguishes a gene that is dark because nobody has looked from one that is dark despite having been studied. Source: PubMed (whole), MULTI-ALIAS sweep of title+abstract: the H37Rv locus tag AND every ortholog identifier (M. bovis Mb…, M. marinum MMAR_…, M. smegmatis MSMEG_…, M. leprae ML…, M. abscessus MAB_…), each hit VERIFIED against the abstract text (word-boundary regex). phase73, 2026-07-13.

Genomic-neighbour overlap (structural caveat) co-directional · 0 % of gene

co-directional overlap: ordinary (e.g. shared stop/start codons in an operon), not the Rv2438A-type artefact P20.1, derived from GFF3 gene coordinates, 2026-08-03.

Binding-pocket screen (P2Rank, geometric prediction) no confident pocket

Read with care. This protein (267 aa) is above the size where the detector reliably differentiates proven enzymes (60.5% confident-pocket rate) from proteins annotated as non-catalytic (18.9%, i.e. 81.1% specificity; P16.3b calibration). Both rates are measured at the threshold this layer applies, best_probability >= 0.70, which is the best separation the detector reaches on that panel (Youden J = 0.416): even at its optimum it misses 39.5% of proven enzymes. 2 candidate pocket(s) were found but none reached confidence (best probability 0.006), which is consistent with a non-catalytic role, though it does not rule out a shallow or non-canonical binding site that this geometric detector misses. (Individual read at this confidence level; the GROUP-level dark-vs-non-catalytic contrast is NOT statistically significant at this size, p=0.285 -- read as modest evidence, not proof, cf. P16.3c.) P16.3e, calibration phase72b_pocket_calibration.py, 2026-08-03.

Conditional expression context (iModulons)

Member of 1 independently-modulated gene set(s): Rv0576 (Rv0576).

iModulon membership (independently-modulated gene sets from a 647-sample RNA-seq compendium): the conditional co-expression context. Co-expression is a regulatory context, NOT a molecular function. Source: iModulonDB / modulome_mtb (Yoo 2022).

CRISPRi vulnerability

Vulnerability index -9.91 (95% CI -11.01 to -8.79). A more negative index = more vulnerable to knockdown (better drug-target quality); indicative threshold VI ≤ -6 = highly vulnerable.

Quantitative CRISPRi knockdown, graded (finer than binary Tn-seq essentiality). Source: CRISPRi vulnerability index (Bosch 2021, supplementary Table S2 'mmc3.xlsx', bulk import via Europe PMC -- supersedes the per-gene pebble.rockefeller.edu scrape of phase46_crispri_vulnerability.py).

Orthologues (reciprocal best hits across mycobacteria)

Reciprocal-best-hit orthologues (DIAMOND) against the Mycobrowser reference proteomes. A missing species is informative: e.g. a gene absent from M. leprae was likely lost in its reductive genome evolution. Locus tags link to Mycobrowser.

Curated reference (UniProt)

Functional vocabulary (eggNOG-mapper, orthology transfer)

Orthology-based transfer (eggNOG 5.0.2, diamond). EC/KO/GO/CAZy are computed annotations, not manual curation; cross-check against the primary literature before treating a specific reaction as established.

Conservation & selection (intra-MTBC, 142 601 strains, snapshot of 2026-09-29)

pN/pS from segregating SNPs (singletons removed) normalised by possible sites. Low pN/pS = purifying selection (a strong signal that a "hypothetical" is a real, constrained gene). A high pN/pS is ambiguous: relaxed constraint or positive selection (drug resistance, antigenic variation) inflate it; e.g. rpoB/katG/pncA score high here for resistance, not loss of function. A clonal disruption (one allele over a clade) suggests lineage pseudogenisation; a convergent one (many independent alleles) is typical of resistance loss-of-function.

Outgroup conservation (beyond the MTBC) Mycobacterium

Two orthogonal outgroup signals. M. canettii (the immediate outgroup) gives a deep-divergence dN/dS (a low value confirms a constrained, real gene; shown as confident only at ≥8 substitutions, else flagged low-power). Genus-wide presence/absence (tblastn vs assembled non-MTBC genomes) places the gene on the ancient-core ↔ MTBC-specific axis: a gene absent even from the closest MTBAP relatives is a candidate MTBC-specific innovation (possible host-adaptation factor, to confirm by synteny). The phylostratum extends that axis outside the genus (tblastn vs 13 reference genomes spanning Mycobacteriaceae → Corynebacteriales → Actinomycetia → outside the phylum): it is the deepest clade in which a homolog is still detected, i.e. a proxy for gene age. Read it with the null model in mind: a shallow (young) stratum can also reflect homology-detection failure for short or fast-evolving ORFs, so it is a descriptive axis, not a proof of novelty.

Essentiality (transposon mutagenesis) ESD — not strictly essential

Genome-wide Himar1 transposon essentiality in H37Rv (DeJesus 2017). An essential call (ES/ESD/GD) is strong, independent evidence that a "hypothetical" locus encodes a functional, selectively required gene — orthogonal to intra-species conservation.

Proteomics (mass spectrometry) detected

Detection by mass spectrometry is direct, experimental evidence that the protein product exists — orthogonal to sequence conservation and to Tn-seq essentiality, and especially decisive for a "hypothetical" locus. Read the dataset count as abundance, not as replication. Measured across this atlas (3,491 detected proteins), the number of datasets a protein appears in tracks its abundance almost monotonically: median 1.2 ppm at 1 dataset of 16, 27 ppm at 10, 392 ppm at 15, 1,325 ppm at 16 — three orders of magnitude. A high count therefore says the protein is abundant more than it says several independent experiments confirmed it. What the count does establish, at any value, is that the product exists.

Predicted localisation (DeepTMHMM + lipobox) no export signal, no TM helix

A verified absence of signals, which is not a compartment. No Sec/Tat signal peptide, no transmembrane helix and no lipobox were found on this sequence. The predictor reports topology and export signals; it does not measure where the protein ends up, so this page draws no cytoplasmic conclusion from it. In M. tuberculosis that reservation is not theoretical: the 12 Esx paralogues of this atlas — EsxA/ESAT-6 and EsxB/CFP-10 among them, the best-established secreted proteins of the species — carry this very call, because substrates of the type VII (ESX) secretion system leave no recognisable signal. « Secreted » and « no signal peptide » are true together here.

Transmembrane topology and signal peptide from DeepTMHMM (deep-learning reference predictor); lipoproteins from a (myco)bacterial lipobox motif. A sequence-based prediction of subcellular context.

Physico-chemical properties (computed, ProtParam)

Computed from the ancestral MTBC0 sequence with the ExPASy ProtParam method (Biopython). Descriptive biophysical context: a positive GRAVY flags a hydrophobic (often membrane) protein, a high instability index (>40) predicts a short in-vitro half-life, an extreme pI hints at compartment or binding partner.

Domains (Pfam, hmmscan --cut_ga)

No Pfam-A domain above the gathering threshold (hmmscan --cut_ga scan completed).

Tentative domain (below the --cut_ga gathering threshold; a low-confidence homology lead, not a firm assignment): Sigma70_r4 (PF04545.23), i-Evalue 8.4e-04, residues 98–121 — Sigma-70, region 4.

Family undetermined, type recognised. 12 distinct Pfam families match the same window (98–121): HTH_10, HTH_11, HTH_17, HTH_20, HTH_23, HTH_24, HTH_AsnC-type, KORA, MarR_2, Rv2175c_wHTH, Sigma70_r4, Sigma70_r4_2. 12 distinct Pfam families overlap the same window (98-121) and 58% of them share the token 'HTH': the REGION and its general TYPE are recognised, the FAMILY is not. Read as 'a HTH-type domain, family undetermined', never as an assignment to the top-scoring family alone. Overlapping Pfam models of one superfamily are NOT independent evidence: their convergence is partly mechanical. Weigh the window, not the count.

HHpred profile-profile: top hits AlpA/excisionase family winged-HTH (PF05930/COG3311), unit B isolated (aa 88-147): Probability 99.28%, E=1.3e-10, best template 8C3T (Wen et al. 2022 NAR) -- mtbc/Rv2516c/, P2.1, >4500x stronger than the full-length search below, Same AlpA/excisionase family, full-length search (alignment window ~89-152): Probability 98.6%, E=4e-07 (this campaign, phase191_hhpred_campaign38_p813.py, 2026-09-01) and Probability 98.64%, E=5.9e-7 (independent earlier run, mtbc/Rv2516c/ P2.1) -- two separate HHpred web submissions converging on the same family, RdfS recombination-directionality factor, rank-2 hit on unit B, E=2.8e-10 (Ramsay et al. 2006 Mol Microbiol; Verdonk et al. 2022 Acta Cryst D) -- same wHTH superfamily, corroborating rather than a distinct alternative. Remote homology search on the deep UniRef30 profile (MPI Toolkit), run for the residual dark set.

3D structure (interactive — Mol*/pdbe-molstar)

No experimental structure is deposited for this gene: showing the AlphaFold DB predicted model (mean pLDDT 80.9, confident), coloured by per-residue confidence (dark blue = very high, orange/red = low — treat low-confidence regions as unreliable, especially termini and loops). Drag to rotate, scroll to zoom.

Structural neighbours (Foldseek on the ESMFold model, exploratory)

ESMFold model confidence: mean pLDDT 86.2 (confident). A confident model makes the fold comparison meaningful.

Best matches against the PDB, ranked by Foldseek homology probability. A high probability / TM-score suggests a shared fold; unless flagged sig (E < 0.01) these are fold hypotheses, not assignments.

Genomic context (neighbours & predicted operon) operon of 2

Neighbours from the H37Rv annotation (- strand). The operon is predicted by co-directional intergenic distance (same strand, gaps ≤50 bp) — a transcription-unit hypothesis, not a mapped TSS. For a "hypothetical", co-transcription with a characterised operon is a concrete functional lead (complements the STRING neighborhood channel below).

Published operon evidence (two independent sources)

Two independent published operon maps, cited alongside the distance heuristic above: an association corroborating or contradicting the heuristic, never proof of shared function. REMap's RNA-seq coverage continuity is the most direct of the three signals; Roback's logistic regression and the intergenic-distance heuristic are statistical proxies.

Transcriptional regulation (signed TRN: ChIP-seq + TFOE)

Regulatory edges from the ISB signed transcriptional regulatory network (TF ChIP-seq binding, Minch 2015 + TF-overexpression response, Rustad 2014). An edge is regulatory evidence (binding and/or expression change), not necessarily direct. For a "hypothetical", membership in a known regulon (e.g. DosR dormancy, PhoP virulence) is a physiological-context lead, not a function.

Condition: Condition-blind by construction. These edges come from transcription-factor OVEREXPRESSION in a single laboratory condition, so a listed regulator is not a claim that it governs this gene in every circumstance. Measured counter-examples exist within this atlas (see Rv2516c, where the DosR regulon collapses under moxifloxacin while the gene itself rises). Read a regulator as 'capable of shifting this gene', and look for the condition before building a hypothesis on it.

Node colour = verdict, dashed = still uncharacterised; edge colour = evidence (green experimental, orange genomic-context, grey co-expression), width ∝ score. Click a partner to open its page; "Explore full network" to walk the graph.

Closest characterised functional partner: Rv2517c (hypothetical protein), high confidence (score 904, all STRING channels except text-mining; the partner is chosen on its genomic-context evidence).

All 3 partner(s) at medium confidence are shown. None rests on an interaction measured in M. tuberculosis itself.

STRING combines evidence channels (neighborhood, fusion, cooccurrence, coexpression, experimental, database, text-mining) into a 0–1000 score. The ctx badge marks edges carried by the genomic-context channels (conserved neighborhood, fusion, phylogenetic co-occurrence), which are independent of orthology and structure and the strongest signal for an unknown gene. The no text-mining column recomputes the score from data alone, so a link that does not depend on the literature is visible. Association is a function hypothesis, not proof: corroborate with the operon context and the primary literature before assigning a function.

Where the experimental evidence comes from, and why it matters here. STRING's experimental channel fuses interactions measured in M. tuberculosis with interactions measured in another organism and transferred to this pair by orthology. The two are not equivalent for this atlas, whose standing rule is that better studied elsewhere is not a function established in M. tuberculosis. Measured on the served network: of the 38 229 partner pairs shown across the atlas, 13 483 partner rows carry a non-zero experimental channel, and only 106 pairs rest on an interaction measured in M. tuberculosis itself — 0.3 % of the network. Those 106 are long-established complexes (ESX-1/ESAT-6, RNA polymerase with CarD and RbpA, the Pup–Mpa proteasome, gyrase, Emb, GarA with PknB and PknG, the Rel modules); no still-uncharacterised gene is among them. The reason is not a gap in this atlas but in the literature: there is almost no experimental interactome of this bacterium, IntAct holding 134 curated protein pairs for a ~4 000-gene proteome. A exp (transferred) badge is therefore a lead, never a measurement, and an absence of badge is not evidence that the pair does not interact.

Evidence

• Legacy H37Rv annotation: hypothetical protein
• MTBC0 PGAP product: hypothetical protein
• Foldseek best: 7b90-assembly1_E Circular permutant of ribosomal protein S6, P54-55 truncated, I (prob 0.99, E=1e-01, TM=0.64)
• (initial automatic assignment: auto-curated by rules from PGAP + Pfam + Foldseek; superseded by hand review)
• Hand review 2026-07-30: converging DNA-binding lead - sub-threshold Pfam Sigma70_r4 (PF04545, res. 98-121, i-E 8.4e-4) + canonical HTH motif at those exact positions + UniProt name 'Helix-turn-helix domain-containing protein'. Non-significant, but supported by three sequence/annotation layers plus structure.
• Hand review 2026-07-30: among the 8 Foldseek hits on the AlphaFold model, MerR-family regulators bound to promoter DNA (3hh0 TM 0.686, 5d8c TM 0.708, 5d90 TM 0.683) OUTSCORE by TM the ribosomal S6 permutants (TM 0.617-0.664) that the automatic summary reported as the single best hit. All non-significant.
• Hand review 2026-07-30: verdict deliberately left 'dark' - the convergence of weak leads orients the experimental plan (profile-profile HHpred, DNA-binding assay) and does not assign a function.
• Hand review 2026-07-30: protparam recomputed after fixing a x100 unit bug in the aliphatic index (phase20_protparam.py); this fiche now carries 84.1 instead of 8408.2.
• Hand review 2026-09-01 (P8.14 resync from mtbc/Rv2516c/, it.20): domain-isolated HHpred (unit B, aa 88-147) gives Probability 99.28%, E=1.3e-10 to the AlpA/excisionase winged-HTH family (PF05930/COG3311) -- >4500x stronger than the same search on the full-length protein (Probability 98.6-98.64%, E=4e-07/5.9e-7). Resolves the DNA-binding lead over the ribosomal-S6 reading; verdict moved dark -> requalified.
• Hand review 2026-09-01: unit B is the most genus-conserved segment of the protein (83.8% identity, 4 independent NTM lineages) and carries none of the gene's 5 essential TnSeq sites (all fall in unit A by count coincidence) -- essentiality of the DNA-binding module itself cannot be assessed by TnSeq (no TA site).

Ancestral MTBC0 protein sequence

>mtbc0_002679|Rv2516c|
MTADWVVTFTFDADPSMETMDAWETQLEGFDALVSRVPGHGIDVTVYAPGDWSVFDALAKMAGEVMPVVQAKSPIAVQIISEPEHRLRAEAFTTPELMSAAEIADELGVSRQRVHQLRSTAGFPAPLADLRGGAVWDAAAVRRFAETWERKPGRPHTGTAKFAYSWAVGPAVGRSGKAPNVRWRVENPDKIRFVLRNIGDDIAEDVEIDLSRIDAITRNVPKKTVIRPGEGLNMVLIAAWGHPLPNQLYVRWAGQDEWAAVPLHPAH