We cloned the intact T4 rHB gene by joining plasmids carrying gene fragments. rIlB was expressed at a low level under control of the lac promoter, and the clone complemented rIIB mutants. We suspect that earlier attempts to clone the intact gene were unsuccessful because of transcription from T4 middle-mode promoters. These promoters are silent early in infection but are recognized when resident on a plasmid in an uninfected cell.
The C‐terminal domain of RNA polymerase II (CTD) acts as a platform to recruit factors that facilitate gene expression, RNA processing, and integrate transcription with the essential cellular processes of DNA repair and histone modification. This domain is composed of a species‐specific number of repeats of the consensus amino acid heptad YSPTSPS, classically numbered as Tyr1‐Ser2‐Pro3‐Thr4‐Ser5‐Pro6‐Ser7. The targeted recruitment of these myriad factors is accomplished by post‐translational modification (PTM) of these heptads, specifically phosphorylation of Tyr1, Ser2, Ser5, and Ser7. Although the contribution of these marks in isolation is extensively studied, the ability of these marks to co‐exist or influence one another has not been rigorously investigated.We take the first steps to characterize “cross‐talk” between phosphorylation marks and reveal a previously unknown role for Tyr1 phosphorylation in diversifying and directing CTD modification events. Tyr1 phosphorylation has been previously implicated in RNA polymerase II stability, antisense and enhancer transcription, anti‐termination, and as an indicator of DNA damage. However, the mechanism through which Tyr1 influences this diverse array of functions is unknown. Using a combination of mass spectrometry, molecular biology, and targeted cellular assays we find that Tyr1 phosphorylation generates a chemically diverse CTD landscape and can influence the specificity of downstream modification enzymes, resulting in novel and uncharacterized marks in the CTD code. We provide direct evidence for combinatorial CTD phosphorylation events, expand the lexicon of CTD coding marks, and demonstrate unexplored mechanisms to precisely control eukaryotic transcription and transcription coupled‐processes.Support or Funding InformationThis work is supported by grants from the National Institutes of Health (R01 GM104896 to Y.J.Z. and R21EB018391 to J.S.B.) and Welch Foundation (F‐1778 to Y.J.Z. and F‐1155 to J.S.B.). Voyager DE‐PROT MALDI MS data was collected in the University of Texas at Austin Proteomics Facility. Funding from the UT System for support of the UT System Proteomics Core Facility Network is gratefully acknowledged.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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