A theoretical study of the mechanism of the nucleotidyl transfer reaction catalyzed by yeast RNA polymerase Ⅱ

The mechanism of the nucleotidyl transfer reaction catalyzed by yeast RNA polymerase I1 has been investigated using molec- ular mechanics and quantum mechanics methods. Molecular dynamics （MD） simulations were carried out using the TIP3 water model and generalized solvent boundary potential （GSBP） by CHARMM based on the X-ray crystal structure. Two models of the ternary elongation complex were constructed based on CHARMM MD calculations. All the species including reactants, transition states, intermediates, and products were optimized using the DFT-PBE method coupled with the basis set DZVP and the auxiliary basis set GEN-A2. Three pathways were explored using the DFT method. The most favorable reaction pathway involves indirect proton migration from the RNA primer 3＇-OH to the oxygen atom of a-phosphate via a solvent water mole- cule, proton rotation from the oxygen atom of a-phosphate to the 13-phosphate side, the RNA primer 3＇-O nucleophilic attack on the a-phosphorus atom, and P-O bond breakage. The corresponding reaction potential profile was obtained. The rate limit- ing step, with a barrier height of 21.5 kcal/mol, is the RNA primer 3＇-0 nucleophilic attack, rather than the commonly consid- ered proton transfer process. A high-resolution crystal structure including crystallographic water molecules is required for fur- ther studies.

How to make the end of a gene,the simple way

Transcription termination of nearly all protein-coding genes in mammals requires 3’end processing by a multiprotein complex that will cleave and polyadenylate the messenger RNA precursor.Because a variety of enzyme complexes intervene,3’end processing was thought to be fundamentally complex and subject to a multitude of regulatory effects.The possibility to select just one out of several polyadenylation sites,in particular,has caused much questioning and speculation.What appear to be separate mechanisms however can be combined into a defined set of rules,allowing for a relatively simple interpretation of 3’end processing.Ultimately,readiness of the terminal exon splice site determines when a transcript reaches the maturity to select a nearby polyadenylation signal.Transcriptional pausing then acts in concert,extending the timeframe during which the transcription complex is close to polyadenylation sites.Since RNA polymerase pausing is governed by the same type of sequences in bacteria and metazoans,mammalian transcription termination resembles its prokaryote counterpart more than generally thought.

Transcriptional pausing induced by ionizing radiation enables the acquisition of radioresistance in nasopharyngeal carcinoma

Lesions on the DNA template can impact transcription via distinct regulatory pathways.Ionizing radiation(IR)as the mainstay modality for many malignancies elicits most of the cytotoxicity by inducing a variety of DNA damages in the genome.How the IR treatment alters the transcription cycle and whether it contributes to the development of radioresistance remain poorly understood.Here,we report an increase in the paused RNA polymerase II(RNAPII),as indicated by the phosphorylation at serine 5 residue of its C-terminal domain,in recurrent nasopharyngeal carcinoma(NPC)patient samples after IR treatment and cultured NPC cells developing IR resistance.Reducing the pool of paused RNAPII by either inhibiting TFIIH-associated CDK7 or stimulating the positive transcription elongation factor b,a CDK9-CycT1 heterodimer,attenuates IR resistance of NPC cells.Interestingly,the poly(ADP-ribosyl)ation of CycT1,which disrupts its phase separation,is elevated in the IR-resistant cells.Mutation of the major poly(ADP-ribosyl)ation sites of CycT1 decreases RNAPII pausing and restores IR sensitivity.Genome-wide chromatin immunoprecipitation followed by sequencing analyses reveal that several genes involved in radiation response and cell cycle control are subject to the regulation imposed by the paused RNAPII.Particularly,we identify the NIMA-related kinase NEK7 under such regulation as a new radioresistancefactor,whose downregulation results in the increased chromosome instability,enabling the development of IR resistance.Overall,our results highlight a novel link between the alteration in the transcription cycle and the acquisition of IR resistance,opening up new opportunities to increase the efficacy of radiotherapy and thwart radioresistance in NpC.

DDB1 prepares brown adipocytes for cold-induced thermogenesis

Brown adipose tissue(BAT)plays a key role in thermogenesis during acute cold exposure.However,it remains unclear how BAT is prepared to rapidly turn on thermogenic genes.Here,we show that damage-specific DNA binding protein 1(DDB1)mediates the rapid transcription of thermogenic genes upon acute cold exposure.Adipose-or BAT-specific Ddb1 knockout mice show severely whitened BAT and significantly decreased expression of thermogenic genes.These mice develop hypothermia when subjected to acute cold exposure at 4℃ and partial lipodystrophy on a high-fat diet due to deficiency in fatty acid oxidation.Mechanistically,DDB1 binds the promoters of Ucp1 and Ppargc1a and recruits positive transcriptional elongation factor b(P-TEFb)to release promoter-proximally paused RNA polymerase II(Pol II),thereby enabling rapid and synchronized transcription of thermogenic genes upon acute cold exposure.Our findings have thus provided a regulatory mechanism of how BAT is prepared to respond to acute cold challenge.