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

Bharati, Akhilendra Pratap; Singh, Neha; Kumar, Vikash; Kashif, Md.; Singh, Amit Kumar; Singh, Priyanka; Singh, Sudhir; Siddiqi, Mohammad Imran; Tripathi, Timir; Akhtar, Maaz

doi:10.1038/srep31294

Cited by 24 publications

(21 citation statements)

References 30 publications

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“…Ser 5 -phosphorylated Pol II has been shown to co-purify with the capping machinery and TFIIH kinase [39]. Recent research has shown that Ser7 phosphorylation is also important for capping: it is necessary for GT Ceg1 association with Pol II in yeast [40].…”

Section: Capping Machinery Functioning Depends On Transcriptionmentioning

confidence: 99%

Interplay of mRNA capping and transcription machineries

et al. 2020

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Early stages of transcription from eukaryotic promoters include two principal events: the capping of newly synthesized mRNA and the transition of RNA polymerase II from the preinitiation complex to the productive elongation state. The capping checkpoint model implies that these events are tightly coupled, which is necessary for ensuring the proper capping of newly synthesized mRNA. Recent findings also show that the capping machinery has a wider effect on transcription and the entire gene expression process. The molecular basis of these phenomena is discussed.

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Section: Capping Machinery Functioning Depends On Transcriptionmentioning

confidence: 99%

Interplay of mRNA capping and transcription machineries

et al. 2020

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“…The E. coli (BL21-DE3) was transformed with the expression construct. The protein was expressed and purified using Ni-NTA column as described somewhere [18,19]. Further purification was done using Superdex TM 75, 10/300GL column on AKTA FPLC (GE Healthcare) pre-calibrated with standard molecular weight markers.…”

Section: Cloning Expression and Purification Of Sus1mentioning

confidence: 99%

An insight into structural plasticity and conformational transitions of transcriptional co-activator Sus1

et al. 2020

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RNA biogenesis and mRNA transport are an intricate process for every eukaryotic cell. SAGA, a transcriptional coactivator and TREX-2 are the two major complexes participate in this process. Sus1 is a transcription export factor and part of both the SAGA and the TREX-2 complex. The competitive exchange of Sus1 molecule between SAGA and TREX-2 complex modulates their function which is credited to structural plasticity of Sus1. Here, we portray the biophysical characterization of Sus1 from S. cerevisiae. The recombinant Sus1 is a α-helical structure which is stable at various pH conditions. We reported the α-helix to βsheet transition at the low pH as well as at high pH. Sus1 showed 50% reduction in the fluorescence intensity at pH-2 as compared to native protein. The fluorescence studies demonstrated the unfolding of tertiary structure of the protein with variation in pH as compared to neutral pH. The same results were obtained in the ANS binding and acrylamide quenching studies. Similarly, the secondary structure of the Sus1 was found to be stable till 55% alcohol concentration while tertiary structure was stable up to 20% alcohol concentration. Further increase in the alcohol concentration destabilizes the secondary as well as tertiary structure. The 300 mM concentration of ammonium sulfate also stabilizes the secondary structure of the protein. The structural characterization of this protein is expected to unfold the process of the transportation of the mRNA with cooperation of different proteins.

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“…Both the novel CDS2 and CDS-Y2 sites are also observed at the same positions in the C. albicans cocrystal structure ( Figures 3A and 8A). In addition, CDS2 residues have been shown to be essential for CTD binding to the S. cerevisiae CE GTase (80). Sequence analysis comparing animal and yeast species shows that the core residues of both CDS2 and CDS-Y2 are functionally conserved across animals and yeasts ( Figure 8B).…”

Section: The Novel Phosphoserine Pocket Cds2 Is Essential For Gtasementioning

confidence: 99%

Identification of novel RNA Polymerase II CTD interaction sites on the mRNA Capping Enzyme

Bage

Almohammed

Cowling

et al. 2020

Preprint

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Recruitment of the mRNA Capping Enzyme (CE/RNGTT) to the site of transcription is essential for the formation of the 5' mRNA cap, which in turn ensures efficient transcription, splicing, polyadenylation, nuclear export and translation of mRNA in eukaryotic cells. The CE is recruited and activated by the Serine-5 phosphorylated carboxyl-terminal domain (CTD) of RNA polymerase II. Through the use of molecular dynamics simulations and enhanced sampling techniques, we provide a systematic and detailed characterisation of the human CE-CTD interface, describing the effect of the CTD phosphorylation state, length and orientation on this interaction. Our computational analyses identify novel CTD interaction sites on the human CE surface and quantify their relative contributions to CTD binding. We also identify differences in the CTD binding conformation when phosphorylated at either the Serine-2 or Serine-5 positions, thus providing insights into how the CE reads the CTD code. The computational findings are then validated by binding and activity assays. These novel CTD interaction sites are compared with cocrystal structures of the CE-CTD complex in different eukaryotic taxa, leading to the conclusion that this interface is considerably more conserved than previous structures have indicated. KeywordsmRNA Capping Enzyme, RNA Polymerase II C-terminal Domain, CTD code, MD simulation, protein-peptide interaction 1 2 Introduction mRNA capping is an essential process required for efficient gene expression and regulation in all eukaryotic organisms (1). The mRNA cap prevents degradation by 5'-exonucleases during transcription and acts as a platform to recruit initiation factors required for splicing, polyadenylation, nuclear export and translation (2-8). mRNA is capped at the 5'-end with an inverted 7methylguanosine moiety. This process occurs in three stages: i) the 5'-end triphosphate is hydrolysed to diphosphate; ii) GMP is covalently linked to the diphosphate 5' end; iii) the guanosine base is methylated at the N7 position (1). In animals the first two stages are performed by a bifunctional protein, the Capping Enzyme (CE/RNGTT), which contains triphosphatase (TPase) and guanylyltransferase (GTase) enzymatic domains separated by a disordered linker (9, 10). The mammalian CE GTase functions independently of the TPase domain (10-12). The final step, N7 methylation of the guanosine base, is performed by RNMT in complex with its activating mini-protein RAM (13,14).The process of mRNA capping is tightly coupled to transcription, occurring during the elongation phase (15,16). At this stage the CE is recruited to the site of transcription by the RNA polymerase II (Pol II) carboxyl-terminal domain (CTD) (17,18). The CTD is located in RPB1, the largest subunit of RNA Pol II, and is composed of a tandem repeated heptad motif with the consensus sequence Y 1 S 2 P 3 T 4 S 5 P 6 S 7 (19,20). This domain is disordered and can be dynamically phosphorylated at several positions to form a highly complex pattern known as the CTD phosphorylat...

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

Cited by 24 publications

References 30 publications

Interplay of mRNA capping and transcription machineries

Interplay of mRNA capping and transcription machineries

An insight into structural plasticity and conformational transitions of transcriptional co-activator Sus1

Identification of novel RNA Polymerase II CTD interaction sites on the mRNA Capping Enzyme

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