Subunit Of Holoenzyme Research Articles

Iron-(Fe3+)-Dependent Reactivation of Telomerase Drives Colorectal Cancers.

Over-consumption of iron-rich red meat and hereditary or genetic iron overload are associated with an increased risk of colorectal carcinogenesis, yet the mechanistic basis of how metal-mediated signaling leads to oncogenesis remains enigmatic. Using fresh colorectal cancer samples we identify Pirin, an iron sensor, that overcomes a rate-limiting step in oncogenesis, by reactivating the dormant human telomerase reverse transcriptase (hTERT) subunit of the telomerase holoenzyme in an iron-(Fe3+)-dependent manner and thereby drives colorectal cancers. Chemical genetic screens combined with isothermal dose-response fingerprinting and mass spectrometry identified a small molecule SP2509 that specifically inhibits Pirin-mediated hTERT reactivation in colorectal cancers by competing with iron-(Fe3+) binding. Our findings, first to document how metal ions reactivate telomerase, provide a molecular mechanism for the well-known association between red meat and increased incidence of colorectal cancers. Small molecules like SP2509 represent a novel modality to target telomerase that acts as a driver of 90% of human cancers and is yet to be targeted in clinic. Significance: We show how iron-(Fe3+) in collusion with genetic factors reactivates telomerase, providing a molecular mechanism for the association between iron overload and increased incidence of colorectal cancers. Although no enzymatic inhibitors of telomerase have entered the clinic, we identify SP2509, a small molecule that targets telomerase reactivation and function in colorectal cancers.

Antagonistic interactions safeguard mitotic propagation of genetic and epigenetic information in zebrafish

The stability of cellular phenotypes in developing organisms depends on error-free transmission of epigenetic and genetic information during mitosis. Methylation of cytosine residues in genomic DNA is a key epigenetic mark that modulates gene expression and prevents genome instability. Here, we report on a genetic test of the relationship between DNA replication and methylation in the context of the developing vertebrate organism instead of cell lines. Our analysis is based on the identification of hypomorphic alleles of dnmt1, encoding the DNA maintenance methylase Dnmt1, and pole1, encoding the catalytic subunit of leading-strand DNA polymerase epsilon holoenzyme (Pole). Homozygous dnmt1 mutants exhibit genome-wide DNA hypomethylation, whereas the pole1 mutation is associated with increased DNA methylation levels. In dnmt1/pole1 double-mutant zebrafish larvae, DNA methylation levels are restored to near normal values, associated with partial rescue of mutant-associated transcriptional changes and phenotypes. Hence, a balancing antagonism between DNA replication and maintenance methylation buffers against replicative errors contributing to the robustness of vertebrate development.

The four subunits of rabbit skeletal muscle lactate dehydrogenase do not exert their catalytic action additively

Oligomeric enzymes containing multiple active sites are usually considered to perform their catalytic action at higher rates when compared with their monomeric counterparts. This implies, in turn, that the activity performed by different holoenzyme subunits features additivity. Nevertheless, the extent of this additivity occurring in holoenzymes is far from being adequately understood. To tackle this point, we used tetrameric rabbit lactate dehydrogenase (rbLDH) as a model system to assay the reduction of pyruvate catalysed by this enzyme at the expense of β-NADH under pre-steady-state conditions. In particular, we observed the kinetics of reactions triggered by concentrations of β-NADH equimolar to 1, 2, 3, or all 4 subunits of the rbLDH holoenzyme, in the presence of an excess of pyruvate. Surprisingly, when the concentration of the limiting reactant exceeded that of a single holoenzyme subunit, we observed a sharp slowdown of the enzyme conformational rearrangements associated to the generation and the release of l-lactate. Furthermore, using a model to interpret the complex kinetics observed under the highest concentration of the limiting reactant, we estimated the diversity of the rates describing the action of the different rbLDH subunits.

TERT Extra-Telomeric Roles: Antioxidant Activity and Mitochondrial Protection

Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase holoenzyme, which adds telomeric DNA repeats on chromosome ends to counteract telomere shortening. In addition, there is evidence of TERT non-canonical functions, among which is an antioxidant role. In order to better investigate this role, we tested the response to X-rays and H2O2 treatment in hTERT-overexpressing human fibroblasts (HF-TERT). We observed in HF-TERT a reduced induction of reactive oxygen species and an increased expression of the proteins involved in the antioxidant defense. Therefore, we also tested a possible role of TERT inside mitochondria. We confirmed TERT mitochondrial localization, which increases after oxidative stress (OS) induced by H2O2 treatment. We next evaluated some mitochondrial markers. The basal mitochondria quantity appeared reduced in HF-TERT compared to normal fibroblasts and an additional reduction was observed after OS; nevertheless, the mitochondrial membrane potential and morphology were better conserved in HF-TERT. Our results suggest a protective function of TERT against OS, also preserving mitochondrial functionality.

Phosphorylation status of Bβ subunit acts as a switch to regulate the function of phosphatase PP2A in ethylene-mediated root growth inhibition.

The various combinations and regulations of different subunits of phosphatase PP2A holoenzymes underlie their functional complexity and importance. However, molecular mechanisms governing the assembly of PP2A complex in response to external or internal signals remain largely unknown, especially in Arabidopsis thaliana. We found that the phosphorylation status of Bβ of PP2A acts as a switch to regulate the activity of PP2A. In the absence of ethylene, phosphorylated Bβ leads to an inactivation of PP2A; the substrate EIR1 remains to be phosphorylated, preventing the EIR1-mediated auxin transport in epidermis, leading to normal root growth. Upon ethylene treatment, the dephosphorylated Bβ mediates the formation of the A2-C4-Bβ protein complex to activate PP2A, resulting in the dephosphorylation of EIR1 to promote auxin transport in epidermis of elongation zone, leading to root growth inhibition. Altogether, our research revealed a novel molecular mechanism by which the dephosphorylation of Bβ subunit switches on PP2A activity to dephosphorylate EIR1 to establish EIR1-mediated auxin transport in the epidermis in elongation zone for root growth inhibition in response to ethylene.

A coronavirus disease 2019–vaccinated patient with phosphoinositide 3-kinase disease with mild illness after severe acute respiratory syndrome coronavirus 2 infection

Phosphoinositide 3-kinase (PI3K) disease is a rare disorder that causes severe impairment of the immune system and the ability to fight both bacterial and viral infections.1 PI3K disease can be caused by a mutation in the PIK3R1 gene. The PIK3R1 gene encodes the regulatory subunit of PI3K holoenzyme, activating the pathway involved in modulation of cell proliferation and growth.2 The PI3K disease is characterized by recurrent ear, nose, throat, and respiratory tract infections that can lead to progressive airway damage.

Vacuolar ATPase subunit F is critical for larval survival in Henosepilachna vigintioctopunctata.

Vacuolar ATPase (vATPase) is an important proton pump in insect tissues including gut and Malpighian tubule. Subunit F, one of the 16 subunits of the vATPase holoenzyme, is not well characterized. Here, we found that two HvvATPaseF isoforms were highly expressed in the hindgut and Malpighian tubules (MT) in the 28-spotted lady-beetle Henosepilachna vigintioctopunctata, an agricultural pest that feeds on Solanaceae and Cucurbitaceae. Knockdown of both HvvATPaseF variants by RNA interference (RNAi) delayed larval growth and negatively affected ecdysis and adult emergence. In the midgut, RNAi treatment resulted in the disappearance of peritrophic membrane, the reduction in the size and the impaired integrity of the gut, which was associated with sparse principle cells and an increase in TUNEL- and EdU-positive cells. Whereas the MT were opaque and the tubule lumens were full of urine in dsegfp-fed larvae, the tubules were clear and the tubule lumens were empty in the dsvATPaseF-fed larvae. HvvATPaseF knockdown was also associated with a decrease in the abundance of the fat body and the levels of glucose, trehalose, triglyceride, total soluble amino acids and proteins, and an increase in glycogen. Consistent with the known effects of sugars on chitin formation, both the expression level of a chitin biosynthesis gene and the thickness of the head capsule cuticle were reduced in the HvvATPaseF-depleted beetles. Our results demonstrated that subunit F plays an essential role in H. vigintioctopunctata development.

Modulation of Phosphoprotein Activity by Phosphorylation Targeting Chimeras (PhosTACs).

Protein phosphorylation, which regulates many critical aspects of cell biology, is dynamically governed by kinases and phosphatases. Many diseases are associated with dysregulated hyperphosphorylation of critical proteins, such as retinoblastoma protein in cancer. Although kinase inhibitors have been widely applied in the clinic, growing evidence of off-target effects and increasing drug resistance prompts the need to develop a new generation of drugs. Here, we propose a proof-of-concept study of phosphorylation targeting chimeras (PhosTACs). Similar to PROTACs in their ability to induce ternary complexes, PhosTACs focus on recruiting a Ser/Thr phosphatase to a phosphosubstrate to mediate its dephosphorylation. However, distinct from PROTACs, PhosTACs can uniquely provide target gain-of-function opportunities to manipulate protein activity. In this study, we applied a chemical biology approach to evaluate the feasibility of PhosTACs by recruiting the scaffold and catalytic subunits of the PP2A holoenzyme to protein substrates such as PDCD4 and FOXO3a for targeted protein dephosphorylation. For FOXO3a, this dephosphorylation resulted in the transcriptional activation of a FOXO3a-responsive reporter gene.

DNA polymerase gamma mutations that impair holoenzyme stability cause catalytic subunit depletion.

Mutations in POLG, encoding POLγA, the catalytic subunit of the mitochondrial DNA polymerase, cause a spectrum of disorders characterized by mtDNA instability. However, the molecular pathogenesis of POLG-related diseases is poorly understood and efficient treatments are missing. Here, we generate the PolgA449T/A449T mouse model, which reproduces the A467T change, the most common human recessive mutation of POLG. We show that the mouse A449T mutation impairs DNA binding and mtDNA synthesis activities of POLγ, leading to a stalling phenotype. Most importantly, the A449T mutation also strongly impairs interactions with POLγB, the accessory subunit of the POLγ holoenzyme. This allows the free POLγA to become a substrate for LONP1 protease degradation, leading to dramatically reduced levels of POLγA in A449T mouse tissues. Therefore, in addition to its role as a processivity factor, POLγB acts to stabilize POLγA and to prevent LONP1-dependent degradation. Notably, we validated this mechanism for other disease-associated mutations affecting the interaction between the two POLγ subunits. We suggest that targeting POLγA turnover can be exploited as a target for the development of future therapies.

Protein farnesylation negatively regulates brassinosteroid signaling via reducing BES1 stability in Arabidopsis thaliana.

Brassinosteroids (BRs) are a group of steroidal phytohormones, playing critical roles in almost all physiological aspects during the life span of a plant. In Arabidopsis, BRs are perceived at the cell surface, triggering a reversible phosphorylation‐based signaling cascade that leads to the activation and nuclear accumulation of a family of transcription factors, represented by BES1 and BZR1. Protein farnesylation is a type of post‐translational modification, functioning in many important cellular processes. Previous studies demonstrated a role of farnesylation in BR biosynthesis via regulating the endoplasmic reticulum localization of a key bassinolide (BL) biosynthetic enzyme BR6ox2. Whether such a process is also involved in BR signaling is not understood. Here, we demonstrate that protein farnesylation is involved in mediating BR signaling in Arabidopsis. A loss‐of‐function mutant of ENHANCED RESPONSE TO ABA 1 (ERA1), encoding a β subunit of the protein farnesyl transferase holoenzyme, can alter the BL sensitivity of bak1‐4 from a reduced to a hypersensitive level. era1 can partially rescue the BR defective phenotype of a heterozygous mutant of bin2‐1, a gain‐of‐function mutant of BIN2 which encodes a negative regulator in the BR signaling. Our genetic and biochemical analyses revealed that ERA1 plays a significant role in regulating the protein stability of BES1.

Multi-omic analysis reveals significantly mutated genes and DDX3X as a sex-specific tumor suppressor in cutaneous melanoma.

The high background tumor mutation burden in cutaneous melanoma limits the ability to identify significantly mutated genes (SMGs) that drive this cancer. To address this, we performed a mutation significance study of over 1,000 melanoma exomes, combined with a multi-omic analysis of 470 cases from The Cancer Genome Atlas. We discovered several SMGs with co-occurring loss-of-heterozygosity and loss-of-function mutations, including PBRM1, PLXNC1 and PRKAR1A, which encodes a protein kinase A holoenzyme subunit. Deconvolution of bulk tumor transcriptomes into cancer, immune and stromal components revealed a melanoma-intrinsic oxidative phosphorylation signature associated with protein kinase A pathway alterations. We also identified SMGs on the X chromosome, including the RNA helicase DDX3X, whose loss-of-function mutations were exclusively observed in males. Finally, we found that tumor mutation burden and immune infiltration contain complementary information on survival of patients with melanoma. In summary, our multi-omic analysis provides insights into melanoma etiology and supports contribution of specific mutations to the sex bias observed in this cancer.

A multi-state model of the CaMKII dodecamer suggests a role for calmodulin in maintenance of autophosphorylation.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) accounts for up to 2 percent of all brain protein and is essential to memory function. CaMKII activity is known to regulate dynamic shifts in the size and signaling strength of neuronal connections, a process known as synaptic plasticity. Increasingly, computational models are used to explore synaptic plasticity and the mechanisms regulating CaMKII activity. Conventional modeling approaches may exclude biophysical detail due to the impractical number of state combinations that arise when explicitly monitoring the conformational changes, ligand binding, and phosphorylation events that occur on each of the CaMKII holoenzyme’s subunits. To manage the combinatorial explosion without necessitating bias or loss in biological accuracy, we use a specialized syntax in the software MCell to create a rule-based model of a twelve-subunit CaMKII holoenzyme. Here we validate the rule-based model against previous experimental measures of CaMKII activity and investigate molecular mechanisms of CaMKII regulation. Specifically, we explore how Ca2+/CaM-binding may both stabilize CaMKII subunit activation and regulate maintenance of CaMKII autophosphorylation. Noting that Ca2+/CaM and protein phosphatases bind CaMKII at nearby or overlapping sites, we compare model scenarios in which Ca2+/CaM and protein phosphatase do or do not structurally exclude each other’s binding to CaMKII. Our results suggest a functional mechanism for the so-called “CaM trapping” phenomenon, wherein Ca2+/CaM may structurally exclude phosphatase binding and thereby prolong CaMKII autophosphorylation. We conclude that structural protection of autophosphorylated CaMKII by Ca2+/CaM may be an important mechanism for regulation of synaptic plasticity.

Fbxl17 is rearranged in breast cancer and loss of its activity leads to increased global O-GlcNAcylation

In cancer, many genes are mutated by genome rearrangement, but our understanding of the functional consequences of this remains rudimentary. Here we report the F-box protein encoded by FBXL17 is disrupted in the region of the gene that encodes its substrate-binding leucine rich repeat (LRR) domain. Truncating Fbxl17 LRRs impaired its association with the other SCF holoenzyme subunits Skp1, Cul1 and Rbx1, and decreased ubiquitination activity. Loss of the LRRs also differentially affected Fbxl17 binding to its targets. Thus, genomic rearrangements in FBXL17 are likely to disrupt SCFFbxl17-regulated networks in cancer cells. To investigate the functional effect of these rearrangements, we performed a yeast two-hybrid screen to identify Fbxl17-interacting proteins. Among the 37 binding partners Uap1, an enzyme involved in O-GlcNAcylation of proteins was identified most frequently. We demonstrate that Fbxl17 binds to UAP1 directly and inhibits its phosphorylation, which we propose regulates UAP1 activity. Knockdown of Fbxl17 expression elevated O-GlcNAcylation in breast cancer cells, arguing for a functional role for Fbxl17 in this metabolic pathway.

SDS22 selectively recognizes and traps metal-deficient inactive PP1

The metalloenzyme protein phosphatase 1 (PP1), which is responsible for ≥50% of all dephosphorylation reactions, is regulated by scores of regulatory proteins, including the highly conserved SDS22 protein. SDS22 has numerous diverse functions, surprisingly acting as both a PP1 inhibitor and as an activator. Here, we integrate cellular, biophysical, and crystallographic studies to address this conundrum. We discovered that SDS22 selectively binds a unique conformation of PP1 that contains a single metal (M2) at its active site, i.e., SDS22 traps metal-deficient inactive PP1. Furthermore, we showed that SDS22 dissociation is accompanied by a second metal (M1) being loaded into PP1, as free metal cannot dissociate the complex and M1-deficient mutants remain constitutively trapped by SDS22. Together, our findings reveal that M1 metal loading and loss are essential for PP1 regulation in cells, which has broad implications for PP1 maturation, activity, and holoenzyme subunit exchange.

PITX1 protein interacts with ZCCHC10 to regulate hTERT mRNA transcription.

Telomerase is a ribonucleoprotein ribonucleic enzyme that is essential for cellular immortalization via elongation of telomere repeat sequences at the end of chromosomes. Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase holoenzyme, is a key regulator of telomerase activity. Telomerase activity, which has been detected in the majority of cancer cells, is accompanied by hTERT expression, suggesting that this enzyme activity contributes to an unlimited replication potential of cancer cells via regulation of telomere length. Thus, hTERT is an attractive target for cancer-specific treatments. We previously reported that pared-like homeodomain 1 (PITX1) is a negative regulator of hTERT through direct binding to the hTERT promoter. However, the mechanism by which the function of PITX1 contributes to transcriptional silencing of the hTERT gene remains to be clarified. Here, we show that PITX1 and zinc finger CCHC-type containing 10 (ZCCHC10) proteins cooperate to facilitate the transcriptional regulation of the hTERT gene by functional studies via FLAG pull-down assay. Co-expression of PITX1 and ZCCHC10 resulted in inhibition of hTERT transcription, in melanoma cell lines, whereas mutate-deletion of homeodomain in PITX1 that interact with ZCCHC10 did not induce similar phenotypes. In addition, ZCCHC10 expression levels showed marked decrease in the majority of melanoma cell lines and tissues. Taken together, these results suggest that ZCCHC10-PITX1 complex is the functional unit that suppresses hTERT transcription, and may play a crucial role as a novel tumor suppressor complex.

Magnaporthe oryzae CK2 Accumulates in Nuclei, Nucleoli, at Septal Pores and Forms a Large Ring Structure in Appressoria, and Is Involved in Rice Blast Pathogenesis.

Magnaporthe oryzae (Mo) is a model pathogen causing rice blast resulting in yield and economic losses world-wide. CK2 is a constitutively active, serine/threonine kinase in eukaryotes, having a wide array of known substrates, and involved in many cellular processes. We investigated the localization and role of MoCK2 during growth and infection. BLAST search for MoCK2 components and targeted deletion of subunits was combined with protein-GFP fusions to investigate localization. We found one CKa and two CKb subunits of the CK2 holoenzyme. Deletion of the catalytic subunit CKa was not possible and might indicate that such deletions are lethal. The CKb subunits could be deleted but they were both necessary for normal growth and pathogenicity. Localization studies showed that the CK2 holoenzyme needed to be intact for normal localization at septal pores and at appressorium penetration pores. Nuclear localization of CKa was however not dependent on the intact CK2 holoenzyme. In appressoria, CK2 formed a large ring perpendicular to the penetration pore and the ring formation was dependent on the presence of all CK2 subunits. The effects on growth and pathogenicity of deletion of the b subunits combined with the localization indicate that CK2 can have important regulatory functions not only in the nucleus/nucleolus but also at fungal specific structures such as septa and appressorial pores.

Differential Expression of Genes for Ubiquitin Ligases in Medulloblastoma Subtypes.

Using publically available datasets on gene expression in medulloblastoma (MB) subtypes, we selected genes for ubiquitin ligases and identified statistically those that best predicted each of the four major MB subgroups as separate disease entities. We identify a gene coding for an ubiquitin ligase, ZNRF3, whose overexpression alone can predict the WNT subgroup for 100% in the Pfister dataset. For the SHH subgroup, we identify a gene for a regulatory subunit of the protein phosphatase 2A (PP2A), PPP2R2C, as the major predictor among the E3 ligases genes. The ubiquitin and ubiquitin-like conjugation database (UUCD) lists PPP2R2C as coding for a Cullin Ring ubiquitin ligase adaptor. For group 3 MBs, the best ubiquitin ligase predictor was PPP2R2B, a gene which codes for another regulatory subunit of the PP2A holoenzyme. For group 4, the best E3 gene predictors were MID2, ZBTB18, and PPP2R2A, which codes for a third PP2A regulatory subunit. Heatmap analysis of the E3 gene data shows that expression of ten genes for ubiquitin ligases can be used to classify MBs into the four major consensus subgroups. This was illustrated by analysis of gene expression of ubiquitin ligases of the Pfister dataset and confirmed in the dataset of Cavalli. We conclude that genes for ubiquitin ligases can be used as genetic markers for MB subtypes and that the proteins coded for by these genes should be investigated as subtype specific therapeutic targets for MB.

Role of Cdc23/Mcm10 in generating the ribonucleotide imprint at the mat1 locus in fission yeast.

The developmental asymmetry of fission yeast daughter cells derives from inheriting ‘older Watson’ versus ‘older Crick’ DNA strand from the parental cell, strands that are complementary but not identical with each other. A novel DNA strand-specific ‘imprint’, installed during DNA replication at the mating-type locus (mat1), imparts competence for cell type inter-conversion to one of the two chromosome replicas. The catalytic subunit of DNA Polymerase α (Polα) has been implicated in the imprinting process. Based on its known biochemical function, Polα might install the mat1 imprint during lagging strand synthesis. The nature of the imprint is not clear: it is either a nick or a ribonucleotide insertion. Our investigations do not support a direct role of Polα in nicking through putative endonuclease domains but confirm its indirect role in installing an alkali-labile moiety as the imprint. While ruling out the role of the primase subunit of Polα holoenzyme, we find that mutations in the Polα-recruitment and putative primase homology domain in Mcm10/Cdc23 abrogate the ribonucleotide imprint formation. These results, while confirming the ribonucleotide nature of the imprint suggest the possibility of a direct role of Mcm10/Cdc23 in installing it in cooperation with Polα and Swi1.

Regulation of the Nuclear PKA Holoenzyme

cAMP and PKA activity is regulated in time and space to ensure proper cellular responses. Recently a novel pool of activatable PKA holoenzyme was discovered to reside in the nucleus, expanding the traditional model of PKA signaling. Based on live‐cell imaging we hypothesize that the regulation of the nuclear PKA holoenzyme is dependent on phosphodiesterases (PDEs) anchored to A‐kinase Anchoring Proteins (AKAPs) which create a local microdomain with a lower concentration of cAMP, thus controlling the activation of anchored nuclear PKA. Using superresolution (pcSOFI) and FRAP techniques we've characterized AKAP95 in the nucleus. Co‐IP experiments using nuclear extracts revealed that both the RIIα subunit of the PKA holoenzyme, and multiple PDE4D isoforms interact with AKAP95, supporting our hypothesis and providing specific candidates to further explore this regulation. Our ICUE3 cAMP biosensor was localized to AKAP95 to measure the levels of cAMP directly around AKAP95 in response to differently localized sources of cAMP. We are able to generate cAMP in different cellular compartments in a dose‐dependent manner by targeting genetically encoded soluble adenylyl cyclase (sAC) and stimulating transfected cells with sodium bicarbonate. When a low concentration of cAMP is generated, only cAMP produced in the nucleus can be detected in the vicinity of AKAP95, whereas cAMP produced at the plasma membrane was not detected within this microdomain. These results support the hypothesis that AKAP95 and PDE4Ds create a microdomain that prevents the activation of scaffolded nuclear PKA holoenzyme except by local cAMP signals. We are still exploring the endogenous source of nuclear PKA activation, but hypothesize that cAMP generated by internalized GPCR signaling at the endosome could activate AKAP95‐tethered PKA.Support or Funding InformationThis work was funded by R01 DK073368.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Structural Implications of Mutations Conferring Rifampin Resistance in Mycobacterium leprae

The rpoB gene encodes the β subunit of RNA polymerase holoenzyme in Mycobacterium leprae (M. leprae). Missense mutations in the rpoB gene were identified as etiological factors for rifampin resistance in leprosy. In the present study, we identified mutations corresponding to rifampin resistance in relapsed leprosy cases from three hospitals in southern India which treat leprosy patients. DNA was extracted from skin biopsies of 35 relapse/multidrug therapy non-respondent leprosy cases, and PCR was performed to amplify the 276 bp rifampin resistance-determining region of the rpoB gene. PCR products were sequenced, and mutations were identified in four out of the 35 cases at codon positions D441Y, D441V, S437L and H476R. The structural and functional effects of these mutations were assessed in the context of three-dimensional comparative models of wild-type and mutant M. leprae RNA polymerase holoenzyme (RNAP), based on the recently solved crystal structures of RNAP of Mycobacterium tuberculosis, containing a synthetic nucleic acid scaffold and rifampin. The resistance mutations were observed to alter the hydrogen-bonding and hydrophobic interactions of rifampin and the 5′ ribonucleotide of the growing RNA transcript. This study demonstrates that rifampin-resistant strains of M. leprae among leprosy patients in southern India are likely to arise from mutations that affect the drug-binding site and stability of RNAP.