Md. Sohail Akhtar

TFIIH Kinase Places Bivalent Marks on the Carboxy-Terminal Domain of RNA Polymerase II

Posttranslational modifications of the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) specify a molecular recognition code that is deciphered by proteins involved in RNA biogenesis. The CTD is comprised of a repeating heptapeptide (Y(1)S(2)P(3)T(4)S(5)P(6)S(7)). Recently, phosphorylation of serine 7 was shown to be important for cotranscriptional processing of two snRNAs in mammalian cells. Here we report that Kin28/Cdk7, a subunit of the evolutionarily conserved TFIIH complex, is a Ser7 kinase. The ability of Kin28/Cdk7 to phosphorylate Ser7 is particularly surprising because this kinase functions at promoters of protein-coding genes, rather than being restricted to promoter-distal regions of snRNA genes. Kin28/Cdk7 is also known to phosphorylate Ser5 residues of the CTD at gene promoters. Taken together, our results implicate the TFIIH kinase in placing bivalent Ser5 and Ser7 marks early in gene transcription. These bivalent CTD marks, in concert with cues within nascent transcripts, specify the cotranscriptional engagement of the relevant RNA processing machinery.

The Bacterial Response Regulator ArcA Uses a Diverse Binding Site Architecture to Regulate Carbon Oxidation Globally

Despite the importance of maintaining redox homeostasis for cellular viability, how cells control redox balance globally is poorly understood. Here we provide new mechanistic insight into how the balance between reduced and oxidized electron carriers is regulated at the level of gene expression by mapping the regulon of the response regulator ArcA from Escherichia coli, which responds to the quinone/quinol redox couple via its membrane-bound sensor kinase, ArcB. Our genome-wide analysis reveals that ArcA reprograms metabolism under anaerobic conditions such that carbon oxidation pathways that recycle redox carriers via respiration are transcriptionally repressed by ArcA. We propose that this strategy favors use of catabolic pathways that recycle redox carriers via fermentation akin to lactate production in mammalian cells. Unexpectedly, bioinformatic analysis of the sequences bound by ArcA in ChIP-seq revealed that most ArcA binding sites contain additional direct repeat elements beyond the two required for binding an ArcA dimer. DNase I footprinting assays suggest that non-canonical arrangements of cis-regulatory modules dictate both the length and concentration-sensitive occupancy of DNA sites. We propose that this plasticity in ArcA binding site architecture provides both an efficient means of encoding binding sites for ArcA, σ70-RNAP and perhaps other transcription factors within the same narrow sequence space and an effective mechanism for global control of carbon metabolism to maintain redox homeostasis.

Unusual Structural Features of the Bacteriophage-associated Hyaluronate Lyase (hylp2)

Hyaluronate lyases are a class of endoglycosaminidase enzymes, which are of considerable complexity and heterogeneity. Their primary function is to degrade hyaluronan and certain other glycosaminoglycans and facilitate the spread of disease. Among hyaluronate lyases, the bacteriophage-associated enzymes are unique as they have the lowest molecular mass, very low amino acid sequence homology with bacterial hyaluronate lyases, and exhibit absolute specificity for one type of glycosaminoglycan, i.e. hyaluronan. Despite such unique characteristics significant details on structural features of these lyases are not available. The Streptococcus pyogenes bacteriophage 10403 contains a gene, hylP2, which encodes for hyaluronate lyase (HylP2) in this organism. HylP2 was cloned, overexpressed, and purified to homogeneity. The recombinant HylP2 exists as a homotrimer of molecular mass about 110 kDa, under physiological conditions. Limited proteolysis and guanidine hydrochloride denaturation studies demonstrated that the N-terminal region of the protein is flexible, whereas the C-terminal portion has a compact conformation. The enzyme shows sequential unfolding, with the N-terminal unfolding first followed by the simultaneous unfolding and dissociation of the stabilized trimeric C-terminal domain. We isolated a functionally active C-terminal fragment (Ser128–Lys337) of the protein that was stabilized in a trimeric configuration. Comparative functional studies with full-length protein, N:C complex, and isolated C-terminal domain demonstrated that the active site of HylP2 is present in the C-terminal portion of the enzyme, and the N-terminal portion modulates the substrate specificity and enzymatic activity of the C-terminal domain.

Insight into the structural flexibility and function of Mycobacterium tuberculosis isocitrate lyase.

Isocitrate lyase (ICL), is a key enzyme of the glyoxylate shunt crucial for the survival of Mycobacterium tuberculosis (Mtb) in macrophages during persistent infection. MtbICL catalyses the first step of this carbon anaplerosis cycle and is considered as a potential anti-tubercular drug target. The MtbICL is a tetramer with 222 symmetry, and each subunit of the enzymeis composed of 14 α-helices and 14 β-strands. We studied the conformational flexibility of the enzyme to get a deeper insight into its stability and function. Our studies show that the mutation of His180, close to the MtbICL signature sequence (K193KCGH197) completely abolishes the oligomeric conformation and function of the enzyme. Molecular dynamics studies suggest that the loss of interaction between His180 and Tyr89 most likely alters the orientation of Tyr89 side chain, thereby causing the movement of helices α6, α12, α13 and α14 in the vicinity and affecting the tetrameric assembly. We further show that the oligomerization of MtbICL is primarily mediated by the inter subunit interactions, and strengthened by the helix swapping of α12-α13 between adjacent subunits. Furthermore, the enzyme activity is influenced by the interactions between the residues of lid region (P411NSSTTALTGSTEEGQFH428) and the loop region (T391KHQREV397). Mutation of glutamates of the lid region to non homologous residues (E423A or E424A) or basic residues (E423K or E424K) inactivates the enzyme, whereas the activity is not much compromised in case of homologous mutations (E423D or E424D).

Mycobacterium tuberculosis persistence in various adipose depots of infected mice and the effect of anti-tubercular therapy.

The adipocytes are one of the non-professional phagocytes postulated to be a haven for Mycobacterium tuberculosis during persistence in the human host. The adipocyte - M. tuberculosis interaction data available to date are ex vivo. The present study was primarily aimed to investigate M. tuberculosis infection of adipocytes in course of infection of mouse model. Using primary murine adipocytes, the study first confirmed the infection and immunomodulation of natural adipocytes by M. tuberculosis. The bacilli could be isolated form visceral, subcutaneous, peri renal and mesenteric adipose depots of immunocompetent mice infected with M. tuberculosis intravenously. The bacilli could be isolated from adipocytes and the stromal vascular fraction, even though the numbers were significantly higher in the latter. The bacterial burden in the adipose depots was comparable to those in lungs in the early phase of infection. But with time, the burden in the adipose depots was either decreased or kept under control, despite the increasing burden in the lungs. Infected mice treated with standard anti tubercular drugs, despite effective elimination of bacterial loads in the lungs, continued to harbour M. tuberculosis in adipose depots at loads similar to untreated mice in the late infection phase.

Isocitrate lyase of Mycobacterium tuberculosis is inhibited by quercetin through binding at N-terminus

Combating tuberculosis requires new therapeutic strategies that not only target the actively dividing bacilli but also the dormant bacilli during persistent infection. Isocitrate lyase (ICL) is a key enzyme of the glyoxylate shunt, crucial for the survival of bacteria in macrophages and mice. MtbICL is considered as one of the potential and attractive drug targets against persistent infection. We report the inhibition of MtbICL by quercetin with IC50 of 3.57 μM. In addition, quercetin strongly inhibited the growth of Mtb H37Rv utilizing acetate, rather than glucose as the sole carbon source, suggesting the inhibition of glyoxylate shunt. Quercetin binds at the N-terminus of MtbICL (Kd - 6.68 μM).

Insights into the Mechanism of Action of Hyaluronate Lyase ROLE OF C-TERMINAL DOMAIN AND Ca2+ IN THE FUNCTIONAL REGULATION OF ENZYME

Hyaluronate lyases (HLs) cleave hyaluronan and certain other chondroitin/chondroitin sulfates. Although native HL from Streptococcus agalactiae is composed of four domains, it finally stabilizes after autocatalytic conversion as a 92-kDa enzyme composed of the N-terminal spacer, middle α-, and C-terminal domains. These three domains are independent folding/unfolding units of the enzyme. Comparative structural and functional studies using the enzyme and its various fragments/domains suggest a relatively insignificant role of the N-terminal spacer domain in the 92-kDa enzyme. Functional studies demonstrate that the α-domain is the catalytic domain. However, independently it has a maximum of only about 10% of the activity of the 92-kDa enzyme, whereas its complex with the C-terminal domain in vitro shows a significant enhancement (about 6-fold) in the activity. It has been previously proposed that the C-terminal domain modulates the enzymatic activity of HLs. In addition, one of the possible roles for calcium ions was suggested to induce conformational changes in the enzyme loops, making HL more suitable for catalysis. However, we observed that calcium ions do not interact with the enzyme, and its role actually is in modulating the hyaluronan conformation and not in the functional regulation of enzyme.

Characterization of hyaluronic acid specific hyaluronate lyase (HylP) from Streptococcus pyogenes.

Streptococcus pyogenes is associated with a wide variety of mucosal and invasive infections that claim human life. The conversion from non pathogenic to toxigenic strain of S. pyogenes are thought to be mediated by bacteriophage infection in several cases. The hyaluronic acid (HA) degrading enzyme Hyaluronate lyase (HL) is proposed to be one of the key bacteriophage-encoded virulence factors. In the present work, HL of S. pyogenes bacteriophage H4489A (HylP) was expressed in Escherichia coli, purified and their structural and functional properties were studied. The enzyme exists in an extended trimeric conformation whose function is influenced by calcium ions. The collagenous Gly-X-Y motif of the enzyme influences stability and interact with calcium ions suggesting its role in the enzyme regulation The HylP shows sequential unfolding through the N-terminal domain. The primary catalytic residues of the enzyme seem to be in the first pocket consisting of Asp170 and Tyr182; however the enzyme activity is considerably reduced with mutation in the second pocket consisting of Glu295 and Tyr298. The catalytic residues span between the regions containing 135-308 amino acids where both the catalytic pocket has a prominent positively charged residue. The net positive potential of the cleft may help in recruiting the negatively charged polymeric HA. Interestingly, unlike other phage HLs, HylP is inhibited by l-ascorbic through non competitive manner.

Recombinant expression, purification and preliminary characterization of the mRNA export factor MEX67 of Saccharomyces cerevisiae

The nuclear export of macromolecules is facilitated by the nuclear pore complexes (NPCs), embedded in the nuclear envelope and consists of multi-protein complexes. MEX67 is one of the nuclear export factor responsible for the transport of the majority of cellular mRNAs from the nucleus to the cytoplasm. The mechanism of mRNA transport through NPCs is unclear due to the unavailability of structures and the known interacting partners of MEX67. The mex67 gene was cloned in pQE30A and was expressed in Escherichia coli. A strategy has been developed to purify the insoluble MEX67 using a nickel affinity column with chelating Sepharose fast flow media, after solubilizing with sodium lauroyl sarcosinate (Sarkosyl). The IMAC purified recombinant MEX67 was further purified using SEC to apparent homogeneity (∼8 mg/L). Following SEC, MEX67 was stable and observed to be a 67 kDa monomeric protein as determined by PAGE and the size exclusion chromatography. The availability of large quantities of the protein will help in its biochemical and biophysical characterization, which may lead to the identification of new interaction partners of MEX67 or MEX67 complex.

The mRNA capping enzyme of Saccharomyces cerevisiae has dual specificity to interact with CTD of RNA Polymerase II

RNA Polymerase II (RNAPII) uniquely possesses an extended carboxy terminal domain (CTD) on its largest subunit, Rpb1, comprising a repetitive Tyr1Ser2Pro3Thr4 Ser5Pro6Ser7 motif with potential phosphorylation sites. The phosphorylation of the CTD serves as a signal for the binding of various transcription regulators for mRNA biogenesis including the mRNA capping complex. In eukaryotes, the 5 prime capping of the nascent transcript is the first detectable mRNA processing event, and is crucial for the productive transcript elongation. The binding of capping enzyme, RNA guanylyltransferases to the transcribing RNAPII is known to be primarily facilitated by the CTD, phosphorylated at Ser5 (Ser5P). Here we report that the Saccharomyces cerevesiae RNA guanylyltransferase (Ceg1) has dual specificity and interacts not only with Ser5P but also with Ser7P of the CTD. The Ser7 of CTD is essential for the unconditional growth and efficient priming of the mRNA capping complex. The Arg159 and Arg185 of Ceg1 are the key residues that interact with the Ser5P, while the Lys175 with Ser7P of CTD. These interactions appear to be in a specific pattern of Ser5PSer7PSer5P in a tri-heptad CTD (YSPTSPPS YSPTSPSP YSPTSPPS) and provide molecular insights into the Ceg1-CTD interaction for mRNA transcription.