Rv2516c Still unknown · low

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 induced in the SOS response to moxifloxacin (Iacobino A, Piccaro G, Pardini M, Fattorini L, Giannoni F, 2021, Microorganisms 9(2):255, doi 10.3390/microorganisms9020255) - combined with the predicted HTH this yields a testable 'DNA-damage-responsive regulator' hypothesis. Note also that Rv2517c is NON-essential (NE, VI +0.54, CI crossing zero) where Rv2516c is essential (VI -9.91): an obligate heterodimer is therefore unlikely, and an AF-Multimer test of the pair needs a positive control to be interpretable.

In the literature (TB corpus sweep) never studied

No TB publication mentions this locus tag in its title or abstract (sweep of a ~330k-abstract PubMed TB corpus). This gene is genuinely unstudied: its darkness reflects absence of investigation, not failure of investigation.

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%; P16.3b calibration). 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, pebble.rockefeller.edu).

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, 145 209 strains)

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. Reproducible detection across several independent datasets (PaxDb) makes the existence claim robust; the integrated abundance places the protein in the proteome's dynamic range.

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 (or not yet scanned).

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.

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).

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 strong physiological-context lead.

Node colour = verdict, dashed = hypothetical; 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.

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 exp badge marks an experimentally-supported partner (measured interaction, experimental/database channel ≥400) as opposed to a purely predicted one — but note that the M. tuberculosis experimental interactome is dominated by a noisy bacterial-two-hybrid screen, so a strong measured link that contradicts the operon/localisation context is likely a false positive. 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.

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)
• (auto-curated by rules from PGAP + Pfam + Foldseek; not hand-reviewed)
• 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.

Ancestral MTBC0 protein sequence

>mtbc0_002679|Rv2516c|
MTADWVVTFTFDADPSMETMDAWETQLEGFDALVSRVPGHGIDVTVYAPGDWSVFDALAKMAGEVMPVVQAKSPIAVQIISEPEHRLRAEAFTTPELMSAAEIADELGVSRQRVHQLRSTAGFPAPLADLRGGAVWDAAAVRRFAETWERKPGRPHTGTAKFAYSWAVGPAVGRSGKAPNVRWRVENPDKIRFVLRNIGDDIAEDVEIDLSRIDAITRNVPKKTVIRPGEGLNMVLIAAWGHPLPNQLYVRWAGQDEWAAVPLHPAH