Transcription by RNA polymerase II and the CTD-chromatin crosstalk.

The epigenetic phenomenon is known to derive the phenotypic variation of an organism through an interconnected cellular network of histone modifications, DNA methylation and RNA regulatory network. Transcription for protein coding genes is a highly regulated process and carried out by a large multi-complex RNA Polymerase II. The carboxy terminal domain (CTD) of the largest subunit of RNA Polymerase II consists of a conserved and highly repetitive heptad sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. The epigenetically modified CTD is thought to selectively bind different protein complexes that participate in mRNA biogenesis and export. The CTD and chromatin appears to have a spatial relationship during the transcription cycle, where the epigenetic modifications of CTD not only influence the state of histone modification but also mediates CTD-chromatin crosstalk. In this mini review, we have surveyed and discussed current developments of RNA Polymerase II CTD and its new emerging crosstalk with chromatin, during the stage specific progression of RNA Polymerase II in transcription cycle. This review is mainly focussed on the insights in budding yeast.

Noncanonical CTD kinases regulate RNA polymerase II in a gene-class-specific manner.

Phosphorylation of the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) governs stage-specific interactions with different cellular machines. The CTD consists of Y1S2P3T4S5P6S7 heptad repeats and sequential phosphorylations of Ser7, Ser5 and Ser2 occur universally at Pol II-transcribed genes. Phosphorylation of Thr4, however, appears to selectively modulate transcription of specific classes of genes. Here, we identify ten new Thr4 kinases from different kinase structural groups. Irreversible chemical inhibition of the most active Thr4 kinase, Hrr25, reveals a novel role for this kinase in transcription termination of specific class of noncoding snoRNA genes. Genome-wide profiles of Hrr25 reveal a selective enrichment at 3' regions of noncoding genes that display termination defects. Importantly, phospho-Thr4 marks placed by Hrr25 are recognized by Rtt103, a key component of the termination machinery. Our results suggest that these uncommon CTD kinases place phospho-Thr4 marks to regulate expression of targeted genes.

Mechanisms of Nucleosome Reorganization by PARP1.

Poly(ADP-ribose) polymerase 1 (PARP1) is an enzyme involved in DNA repair, chromatin organization and transcription. During transcription initiation, PARP1 interacts with gene promoters where it binds to nucleosomes, replaces linker histone H1 and participates in gene regulation. However, the mechanisms of PARP1-nucleosome interaction remain unknown. Here, using spFRET microscopy, molecular dynamics and biochemical approaches we identified several different PARP1-nucleosome complexes and two types of PARP1 binding to mononucleosomes: at DNA ends and end-independent. Two or three molecules of PARP1 can bind to a nucleosome depending on the presence of linker DNA and can induce reorganization of the entire nucleosome that is independent of catalytic activity of PARP1. Nucleosome reorganization depends upon binding of PARP1 to nucleosomal DNA, likely near the binding site of linker histone H1. The data suggest that PARP1 can induce the formation of an alternative nucleosome state that is likely involved in gene regulation and DNA repair.

Cdc15 Phosphorylates the C-terminal Domain of RNA Polymerase II for Transcription during Mitosis.

In eukaryotes, the basal transcription in interphase is orchestrated through the regulation by kinases (Kin28, Bur1, and Ctk1) and phosphatases (Ssu72, Rtr1, and Fcp1), which act through the post-translational modification of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. The CTD comprises the repeated Tyr-Ser-Pro-Thr-Ser-Pro-Ser motif with potential epigenetic modification sites. Despite the observation of transcription and periodic expression of genes during mitosis with entailing CTD phosphorylation and dephosphorylation, the associated CTD specific kinase(s) and its role in transcription remains unknown. Here we have identified Cdc15 as a potential kinase phosphorylating Ser-2 and Ser-5 of CTD for transcription during mitosis in the budding yeast. The phosphorylation of CTD by Cdc15 is independent of any prior Ser phosphorylation(s). The inactivation of Cdc15 causes reduction of global CTD phosphorylation during mitosis and affects the expression of genes whose transcript levels peak during mitosis. Cdc15 also influences the complete transcription of clb2 gene and phosphorylates Ser-5 at the promoter and Ser-2 toward the 3' end of the gene. The observation that Cdc15 could phosphorylate Ser-5, as well as Ser-2, during transcription in mitosis is in contrast to the phosphorylation marks put by the kinases in interphase (G1, S, and G2), where Cdck7/Kin28 phosphorylates Ser-5 at promoter and Bur1/Ctk1 phosphorylates Ser-2 at the 3' end of the genes.

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.

Dual targeting of MDM2 with a novel small-molecule inhibitor overcomes TRAIL resistance in cancer.

Mouse double minute 2 (MDM2) protein functionally inactivates the tumor suppressor p53 in human cancer. Conventional MDM2 inhibitors provide limited clinical application as they interfere only with the MDM2-p53 interaction to release p53 from MDM2 sequestration but do not prevent activated p53 from transcriptionally inducing MDM2 expression. Here, we report a rationally synthesized chalcone-based pyrido[ b ]indole, CPI-7c, as a unique small-molecule inhibitor of MDM2, which not only inhibited MDM2-p53 interaction but also promoted MDM2 degradation. CPI-7c bound to both RING and N-terminal domains of MDM2 to promote its ubiquitin-mediated degradation and p53 stabilization. CPI-7c-induced p53 directly recruited to the promoters of DR4 and DR5 genes and enhanced their expression, resulting in sensitization of TNF-related apoptosis-inducing ligand (TRAIL)-resistant cancer cells toward TRAIL-induced apoptosis. Collectively, we identified CPI-7c as a novel small-molecule inhibitor of MDM2 with a unique two-prong mechanism of action that sensitized TRAIL-resistant cancer cells to apoptosis by modulating the MDM2-p53-DR4/DR5 pathway.

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, σ(70)-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.

The prophage-encoded hyaluronate lyase has broad substrate specificity and is regulated by the N-terminal domain.

Streptococcus equi is the causative agent of the highly contagious disease "strangles" in equines and zoonotic meningitis in human. Spreading of infection in host tissues is thought to be facilitated by the bacterial gene encoded extracellular hyaluronate lyase (HL), which degrades hyaluronan (HA), chondroitin 6-sulfate, and dermatan sulfate of the extracellular matrix). The clinical strain S. equi 4047 however, lacks a functional extracellular HL. The prophages of S. equi and other streptococci encode intracellular HLs which are reported to partially degrade HA and do not cleave any other glycosaminoglycans. The phage HLs are thus thought to play a role limited to the penetration of streptococcal HA capsules, facilitating bacterial lysogenization and not in the bacterial pathogenesis. Here we systematically looked into the structure-function relationship of S. equi 4047 phage HL. Although HA is the preferred substrate, this HL has weak activity toward chondroitin 6-sulfate and dermatan sulfate and can completely degrade all of them. Even though the catalytic triple-stranded ß-helix domain of phage HL is functionally independent, its catalytic efficiency and specificity is influenced by the N-terminal domain. The phage HL also interacts with human transmembrane glycoprotein CD44. The above results suggest that the streptococci can use phage HLs to degrade glycosaminoglycans of the extracellular matrix for spreading virulence factors and toxins while utilizing the disaccharides as a nutrient source for proliferation at the site of infection.

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 Ser7 of RNA Pol II-CTD influences the recruitment of Cdc73 for mRNA transcription.

The carboxyl terminal domain of the largest subunit of eukaryotic RNA polymerase II (RNAPII) consists of highly conserved tandem repeats of Tyr1Ser2Pro3Thr4Ser5Pro6Ser7, referred as CTD. The CTD undergoes posttranslational modifications where the interplay of kinases imparts specific CTD phosphorylations, recognized by regulatory proteins that help in the mRNA transcription. Here, the Ser5 phosphorylation (Ser5P) remains high during the transcription initiation, followed by the Ser2P which peaks towards the termination and the Ser7P remains high throughout the transcription process. The Paf1 elongation complex (Paf1C) through its Cdc73 subunit is recruited to the phosphorylated CTD and play active role during different stages of mRNA transcription. We show that the CTD binding domain of Cdc73 is an independent folding unit which interacts with the hyper phosphorylated CTD. The 500 ns MD simulation studies further identified the binding interface and the pattern of CTD phosphorylation involved in the interaction with Cdc73. The possible key residues were mutated and the subsequent pull down analysis suggests that the phosphorylated Ser2, Ser5 and Ser7 of the tandem CTD heptads interact respectively with Arg310, Arg268 and Arg300 of Cdc73. Our finding provides new insight for Cdc73 function during mRNA transcription.

The Saccharomyces cerevisiae SR protein Npl3 interacts with hyperphosphorylated CTD of RNA Polymerase II.

The catalytic subunit of RNA Polymerase II contains a highly conserved carboxy terminal domain (CTD) composed of multiple tandem heptad sequence Tyr1Ser2Pro3Thr4Ser5Pro6Ser7. The non-proline residues in CTD undergo posttranslational modifications, with Ser5 phosphorylation (Ser5P) predominating at the start of the transcription cycle and Ser2P at the end, while other phosphorylation levels are high all throughout. The differentially phosphorylated CTD is recognized by regulatory proteins, helpful during mRNA transcription and export. One such protein Npl3 is composed of two RNA binding domains and a C-terminus RGG/SR domain. The Ser411 of Npl3 is reported to make direct contact with Ser2P of CTD for its recruitment and function, while the Npl3 lacking of C-terminal 25 amino acids (Npl3Δ389-414) showed no apparent defects in mRNA synthesis. Here, we report that the RNA binding domains of Npl3 are separate folding units and interact also with the CTD. The interaction between Npl3 and CTD appears to involve not just Ser2P, but also the Ser5P and Ser7P. The Arg126 of the first RNA binding domain interacts with Ser2P whereas the Arg235 of the second RNA binding domain interacts with either Ser7P or Ser5P of another heptad. The finding provides new insight of Npl3 function for mRNA transcription.

An Unusual Effect of Hofmeister Series Salts on the Stability of Toxoplasma gondii Ferredoxin NADP+ Reductase.

BACKGROUND: The ionic interactions play an important role in the stabilization of the native conformation of proteins. Toxoplasma gondii Ferredoxin NADP+ Reductase (TgFNR) remains stable at pH 4.0. However, such modulation of ionic interactions leads to compaction and non-cooperativity in its folding. OBJECTIVE: To gain insights into the role of ionic interactions in the modulation of structure and thermodynamic stability of TgFNR. METHODS: Protein preparations, circular dichroism and fluorescence spectroscopy were used to determine salt-induced changes in the structure and stability of TgFNR. RESULTS: The kosmotropic salts (sodium fluoride and sodium sulphate) appear to induce the biphasic response on the structure and stability of TgFNR. At pH about 4.0, the addition of low concentrations of kosmotropic salts significantly perturbs the existing native-like secondary structure of TgFNR, whereas higher quantities of salt reversed the denaturing impact. This is a one-of-a-kind situation we are unaware of in any other protein. The urea-induced unfolding of TgFNR in the presence of a low dose of salt (100 mM) drastically affected the protein's thermodynamic stability at neutral pH. The increased salt concentrations, on the other hand, reversed the destabilizing effect. CONCLUSION: Our findings imply that electrostatic interactions are exceptionally significant for the TgFNR stability, however, render highly unusual behavior of Hofmeister series salts, indicating a possible crucial role of salt bridges in the stabilization of different conformations of the protein.

Recombinant expression and biophysical characterization of Mrt4 protein that involved in mRNA turnover and ribosome assembly from Saccharomyces cerevisiae.

The mRNA turnover and ribosome assembly are facilitated by Mrt4 protein from Saccharomyces cerevisiae. In present study, we are reporting the cloning, expression and homogeneous purification of recombinant Mrt4. Mrt4 is a 236-amino-acid-long nuclear protein that plays a very crucial role in mRNA turnover and ribosome assembly during the translation process. mrt4 gene was amplified by polymerase chain reaction and cloned in expression vector pET23a (+) under the bacteriophage T7-inducible promoter and lac operator. Furthermore, protein was purified to homogeneity using immobilized metal affinity chromatography (IMAC) and its homogeneous purification was further validated by immunoblotting with anti-His antibody. The far-UV CD spectra represent that Mrt4 has a typical α helix with characteristic negative minima at 222 and 208 nm. At physiological pH, the fluorescence spectra and CD spectra showed properly folded tertiary and secondary structures of Mrt4, respectively. Saccharomyces Mrt4 protein possesses putative bipartite NLS (nuclear localization signal) at the N-terminal part followed by two well-conserved domains, rRNA-binding domains and translation factor (TF) binding domain. PIPSA analysis evaluates electrostatic interaction properties of proteins and concluded that Mrt4 protein can be used as a fingerprint for classifying Mrt4-like mRNA turnover protein from various species. The availability of an ample amount of protein may help in its biochemical and biophysical characterization, crystallization and identification of new interacting partners of Mrt4.

Proteome analysis of Saccharomyces cerevisiae after methyl methane sulfonate (MMS) treatment.

The treatment of methyl methane sulfonate (MMS) increases sensitivity to the DNA damage which, further leads to the cell death followed by a cell cycle delay. Delay in the cell cycle is because of the change in global transcription regulation which results into proteome change. There are several microarray studies on the transcriptome changes after MMS treatment, but very few studies are reported related to proteome change. The proteome analysis in this report identified subgroups of proteins, belonging to known cell cycle regulators, metabolic pathways and protein folding. About 53 proteins were identified by MS/MS and found that 36 of them were induced, 10 were repressed and few of them showed insignificant change. Our results indicated the change in the interactome as well as phosphorylation status of carboxy terminal domain (CTD) of RNA Polymerase II (RNAP-II) after MMS treatment. The RNAP-II complex was affinity purified and ~1640 peptides were identified using nano LC/MS corresponding to 27 interacting proteins along with the twelve RNAP-II subunit. These identified proteins participated in the repair of the damage, changes the function of the main energetic pathways and the carbon flux in various end products. The main metabolic enzymes in the glycolysis, pyruvate phosphate and amino acid biosynthesis pathways showed significant change. Our results indicate that DNA damage is somehow related to these pathways and is co-regulated simultaneously.

Rv1717 Is a Cell Wall - Associated ß-Galactosidase of Mycobacterium tuberculosis That Is Involved in Biofilm Dispersion.

Understanding the function of conserved hypothetical protein (CHP)s expressed by a pathogen in the infected host can lead to better understanding of its pathogenesis. The present work describes the functional characterization of a CHP, Rv1717 of Mycobacterium tuberculosis (Mtb). Rv1717 has been previously reported to be upregulated in TB patient lungs. Rv1717 belongs to the cupin superfamily of functionally diverse proteins, several of them being carbohydrate handling proteins. Bioinformatic analysis of the amino acid sequence revealed similarity to glycosyl hydrolases. Enzymatic studies with recombinant Rv1717 purified from Escherichia coli showed that the protein is a ß-D-galactosidase specific for pyranose form rather than the furanose form. We expressed the protein in Mycobacterium smegmatis (Msm), which lacks its ortholog. In Msm Rv1717 , the protein was found to localize to the cell wall (CW) with a preference to the poles. Msm Rv1717 showed significant changes in colony morphology and cell surface properties. Most striking observation was its unusual Congo red colony morphotype, reduced ability to form biofilms, pellicles and autoagglutinate. Exogenous Rv1717 not only prevented biofilm formation in Msm, but also degraded preformed biofilms, suggesting that its substrate likely exists in the exopolysaccharides of the biofilm matrix. Presence of galactose in the extracellular polymeric substance (EPS) has not been reported before and hence we used the galactose-specific Wisteria floribunda lectin (WFL) to test the same. The lectin extensively bound to Msm and Mtb EPS, but not the bacterium per se. Purified Rv1717 also hydrolyzed exopolysaccharides extracted from Msm biofilm. Eventually, to decipher its role in Mtb, we downregulated its expression and demonstrate that the strain is unable to disperse from in vitro biofilms, unlike the wild type. Biofilms exposed to carbon starvation showed a sudden upregulation of Rv1717 transcripts supporting the potential role of Rv1717 in Mtb dispersing from a deteriorating biofilm.

New insight on 8-anilino-1-naphthalene sulfonic acid interaction with TgFNR for hydrophobic exposure analysis.

The exposed hydrophobic patches of protein are widely detected through the binding by the fluorescent probes such as 1-anilino-8-naphthalene sulfonate (ANS), Nile Red (NR) and 1-(N-phenylamino) naphthalene, N-(1-Naphthyl) aniline (1NPN). Interestingly, at pH4, where the Toxoplasma gondii Ferredoxin-NADP(+) reductase (TgFNR) is stable, an exclusive binding and fluorescence emission was observed for ANS. To understand the underlying difference in the binding of ANS, NR and 1NPN; their effect on the protein structure was studied in detail. ANS was found to interact with TgFNR via electrostatic as well as hydrophobic interactions at pH4. NR and 1NPN did not show any such binding to TgFNR in the similar conditions, however showed strong hydrophobic interaction in the presence of NaCl or DSS (2, 2-dimethyl-2-silapentane-5-sulfonate). The subsequent structural studies suggest that ANS, NaCl and DSS induced partial unfolding of TgFNR by modulating ionic interactions of the enzyme, leading to the exposure of buried hydrophobic patches amicable for the binding by NR and 1NPN. The induced unfolding of TgFNR by ANS is unique and thus cautions to use the fluorescent dye as simple indicator to probe the exposed hydrophobic patches of the protein or its folding intermediates.

Alternate pathway to ascorbate induced inhibition of Mycobacterium tuberculosis.

Ascorbate has been demonstrated to interfere with the growth of Mycobacterium tuberculosis. It scavenges oxygen in the culture medium to induce dormancy of M. tuberculosis. It kills the mycobacteria by generating reactive oxygen intermediates via iron mediated Fenton reactions. In this study, we observed that ascorbate can inhibit M. tuberculosis isocitrate lyase (MtbICL) with an IC50 of 2.15 mΜ. MtbICL is an essential enzyme for the survival of M. tuberculosis under dormancy. We studied the effect of ascorbate on the growth of M. tuberculosis H37Rv metabolizing through citric acid cycle or glyoxylate cycle with glucose or acetate respectively as the sole carbon source. It was observed that 4 mM ascorbate inhibited ∼89% of the growth in glucose medium, which was confirmed to be mediated by Fenton reaction, as the inhibition was significantly lesser (61%) under low iron condition. On the other hand, in acetate medium, ∼97% of the growth was inhibited and the inhibition was uninfluenced by the iron levels. 3-nitropropionate, a known inhibitor of MtbICL, was seen to cause significantly higher inhibition in the acetate medium than in the glucose medium; however it was indifferent to iron levels in either medium. Molecular docking and dynamic simulation studies confirmed stable binding of ascorbate to MtbICL leading to its inhibition. These observations suggest an additional pathway for ascorbate induced inhibition of M. tuberculosis through inhibition of glyoxylate cycle. Since human immune cells can accumulate ascorbate in millimolar concentrations, the in vitro activity range (1-4 mM) of ascorbate against M. tuberculosis could be extrapolated in vivo. Our result supports the possible benefits of adding high vitamin C diet in TB-treated patients.

Salt-regulated reversible fibrillation of Mycobacterium tuberculosis isocitrate lyase: Concurrent restoration of structure and activity.

Protein fibrillation is associated with a number of neurodegenerative diseases. Nevertheless, several proteins not related to disease can also form fibrils in vitro under specific conditions. In the present study, we demonstrate the reversible fibrillation of a globular protein that is modulated by salt under physiological pH. Mycobacterium tuberculosis Isocitrate lyase (MtbICL) is a crucial enzyme involved in the glyoxylate shunt and a potential drug target against M. tuberculosis infection. Under physiological pH, the enzyme self-assembles into a fibrillar structure in the absence of salt in vitro. The mature fibrillar structure of MtbICL is dynamic and restores its tetrameric structure as well as activity with the addition of salt. The kinetics of fibril formation was investigated spectroscopically using 8-Anilinonaphthalene-1-sulfonic acid (ANS). Further, Transmission electron microscopy (TEM) and Atomic force microscopy (AFM) imaging also confirmed the formation of elongated fibrils in the absence of salt. The results indicate the balance between stabilizing forces and the localized electrostatic repulsions destabilizing the tetrameric MtbICL is adjusted via ion shielding. Our result is in congruence of the hypothesis that amyloid formation is an intrinsic property of most, if not all natural proteins under an appropriate set of conditions.

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.

The C-terminus hot spot region helps in the fibril formation of bacteriophage-associated hyaluronate lyase (HylP2).

The bacteriophage encoded hyaluronate lyases (HylP and HylP2) degrade hyaluronan and other glycosaminoglycans. HylP2 forms a functional fibril under acidic conditions in which its N-terminus is proposed to form the fibrillar core, leading to nucleation and acceleration of fibril formation. Here we report the presence of a hot spot region (A144GVVVY149) towards the carboxy terminus of HylP2, essential for the acceleration of fibril formation. The 'hot spot' is observed to be inherently mutated for valines (A178AMVMY183) in case of HylP. The N- terminal swapped chimeras between these phage HLs ((N)HylP2(C)HylP and (N)HylP(C)HylP2) or HylP did not form fibrils at acidic pH. However, seeding of prefibrils of HylP2 recompensed nucleation and led to fibrillation in (N)HylP(C)HylP2. The V147A mutation in the 'hot spot' region abolished fibril formation in HylP2. The M179V and M181V double mutations in the 'hot spot' region of HylP led to fibrillation with the seeding of prefibrils. It appears that fibrillation in HylP2 even though is initiated by the N-terminus, is accelerated by the conserved 'hot spot' region in the C-terminus. A collagenous (Gly-X-Y)10 motif in the N-terminus and a mutated 'hot spot' region in the C-terminus of HylP affect fibrillar nucleation and acceleration respectively.