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  • Mapping The Phosphorylation of The Human RNA Polymerase II Reveals That The Identity

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    Luís Miguel
    14 vizualizări18 pagini
    This document summarizes the results of a study mapping phosphorylation of the human RNA polymerase II carboxyl-terminal domain (CTD). The study found that the identity and modification of the seventh residue of heptad repeats directs Tyr1 phosphorylation. Specifically, negatively charged residues at position 7 favored Tyr1 phosphorylation, while positively charged or polar residues disfavored it. Tables of mass spectrometry data are provided supporting the identification of phosphorylated Tyr1 residues on various CTD constructs.

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    Mapping the phosphorylation of the human RNA polymerase II reveals that the identity and modification of the seventh residues of heptad repeats direct Tyr1 phosphorylation

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    Mapping the Phosphorylation of the Human RNA Polymerase II Reveals That the Identity

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    This document summarizes the results of a study mapping phosphorylation of the human RNA polymerase II carboxyl-terminal domain (CTD). The study found that the identity and modification of the seventh residue of heptad repeats directs Tyr1 phosphorylation. Specifically, negatively charged residues at position 7 favored Tyr1 phosphorylation, while positively charged or polar residues disfavored it. Tables of mass spectrometry data are provided supporting the identification of phosphorylated Tyr1 residues on various CTD constructs.

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    © All Rights Reserved
    Descărcați ca PDF, TXT sau citiți online pe Scribd
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    14 vizualizări18 pagini

    Mapping The Phosphorylation of The Human RNA Polymerase II Reveals That The Identity

    Încărcat de

    Luís Miguel
    This document summarizes the results of a study mapping phosphorylation of the human RNA polymerase II carboxyl-terminal domain (CTD). The study found that the identity and modification of the seventh residue of heptad repeats directs Tyr1 phosphorylation. Specifically, negatively charged residues at position 7 favored Tyr1 phosphorylation, while positively charged or polar residues disfavored it. Tables of mass spectrometry data are provided supporting the identification of phosphorylated Tyr1 residues on various CTD constructs.

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    © All Rights Reserved
    Descărcați ca PDF, TXT sau citiți online pe Scribd
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    din 18

    Mapping the phosphorylation of the human RNA polymerase II reveals that the identity and

    modification of the seventh residues of heptad repeats direct Tyr1 phosphorylation

    Nathaniel T. Burkholder 1*, Sarah N. Sipe 2*, Edwin E. Escobar 2, Mukesh Kumar 1, Seema

    Irani 1, Wanjie Yang 1, Haoyi Yu 1, Wendy M. Matthews 1, Jennifer S. Brodbelt 2,# and Yan

    Zhang 1,3 #

    SUPPORTING INFORMATION

    Table S1. CTD construct sequences.

    Table S2. Identified ions in UVPD-MS analysis of singly phosphorylated peaks from ABL1
    treated, trypsin digested 26xDistal CTD substrate.

    Table S3. Identified ions in UVPD-MS analysis of singly phosphorylated peaks from ABL1
    treated 3xWT, 3xS7E Mid and Sp, and 3xS2E Mid substrates.

    Table S4. Identified ions in UVPD-MS analysis of singly phosphorylated peaks from ABL1
    treated, GluC digested 26xS7E Sp CTD substrate.

    Table S5. Identified ions in UVPD-MS analysis of singly phosphorylated peaks from ABL1
    treated 3xS7R Sp, Arg1810, and Cit1810 substrates.

    Table S6. Identified ions in UVPD-MS analysis of singly phosphorylated peaks from ABL1
    treated 3xS7K Sp and 3xS7Q Mid/Sp substrates.

    1
    Table S1. CTD construct sequences. 6xHis-GST sequence up to the 3C cleavage site is
    included in the distal CTD sequence, but is written in short hand for all sequences thereafter as
    “6xHis-GST - “. 3C cleavage site is underlined and the cleavage site is indicated with a dash
    (LEVLFQ/GP). Ser7 mutations are highlighted based on residue character (red – negatively
    charged, purple – positively charged, green – polar, yellow – nonpolar).

    CTD Protein/Peptide Sequence

    6xHis-GST-human distal 26x CTD MHHHHHHSSMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGD


    KWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKE
    RAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLC
    HKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIP
    QIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSSSLEVLFQ/GPGSGMS
    PTSPNYTPTSPNYSPTSPSYSPTSPSYSPTSPSYSPSSPRYTPQSPTY
    TPSSPSYSPSSPSYSPTSPKYTPTSPSYSPSSPEYTPTSPKYSPTSPKY
    SPTSPKYSPTSPTYSPTTPKYSPTSPTYSPTSPVYTPTSPKYSPTSPTY
    SPTSPKYSPTSPTYSPTSPKGSTYSPTSPGYSPTSPTYSLTSPAISPDD
    SDEEN

    6xHis-GST-yeast 26x CTD 6xHis-GST -


    GPGSGMGFGVSSPGFSPTSPTYSPTSPAYSPTSPSYSPTSPSYSPTSP
    SYSPTSPSYSPTSPSYSPTSPSYSPTSPSYSPTSPSYSPTSPSYSPTSP
    SYSPTSPSYSPTSPSYSPTSPSYSPTSPSYSPTSPAYSPTSPSYSPTSP
    SYSPTSPSYSPTSPSYSPTSPNYSPTSPSYSPTSPGYSPGSPAYSPKQ
    DEQKHNENENSR

    6xHis-GST-yeast 26x S7E CTD 6xHis-GST -


    GPGSGMGFGVSSPGFSPTSPEYSPTSPEYSPTSPEYSPTSPEYSPTS
    PEYSPTSPEYSPTSPEYSPTSPEYSPTSPEYSPTSPEYSPTSPEYSPTS
    PEYSPTSPEYSPTSPEYSPTSPEYSPTSPEYSPTSPEYSPTSPEYSPTS
    PEYSPTSPEYSPTSPEYSPTSPEYSPTSPEYSPTSPEYSPGSPEYSPK
    QDEQKHNENENSR

    6xHis-GST-yeast 26x S7E Sp CTD 6xHis-GST -


    GPGSGMGFGVSSPGFSPTSPEYSPTSPAYSPTSPEYSPTSPSYSPTS
    PEYSPTSPSYSPTSPEYSPTSPSYSPTSPEYSPTSPSYSPTSPEYSPTS
    PSYSPTSPEYSPTSPSYSPTSPEYSPTSPSYSPTSPEYSPTSPSYSPTS
    PEYSPTSPSYSPTSPEYSPTSPNYSPTSPEYSPTSPGYSPGSPEYSPK
    QDEQKHNENENSR

    6xHis-GST-3xWT 6xHis-GST - GPGSGMSPSYSPTSPSYSPTSPSYSPT

    6xHis-GST-3xS7E Sp 6xHis-GST - GPGSGMSPEYSPTSPSYSPTSPEYSPT

    6xHis-GST-3xS7E Mid 6xHis-GST - GPGSGMSPSYSPTSPEYSPTSPSYSPT

    2
    6xHis-GST-3xS7R Sp 6xHis-GST - GPGSGMSPRYSPTSPSYSPTSPRYSPT

    6xHis-GST-3xS7R Mid 6xHis-GST - GPGSGMSPSYSPTSPRYSPTSPSYSPT

    6xHis-GST-3xS7K Sp 6xHis-GST - GPGSGMSPKYSPTSPSYSPTSPKYSPT

    6xHis-GST-3xS7K Mid 6xHis-GST - GPGSGMSPSYSPTSPKYSPTSPSYSPT

    6xHis-GST-3xS7Q Sp 6xHis-GST - GPGSGMSPQYSPTSPSYSPTSPQYSPT

    6xHis-GST-3xS7Q Mid 6xHis-GST – GPGSGMSPSYSPTSPQYSPTSPSYSPT

    Distal CTD 1801-1822 Arg1810 peptide H-SPSYSPSSPRYTPQSPTYTPSS-OH

    Distal CTD 1801-1822 Cit1810 peptide H-SPSYSPSSPCitYTPQSPTYTPSS-OH

    3
    Table S2. Identified ions in UVPD-MS analysis of singly phosphorylated peaks from ABL1
    treated, trypsin digested 26xDistal CTD substrate (Fig. 1B-G). The phosphorylated Tyr1 sites
    were shown in blue boxes.

    4
    5
    6
    7
    Table S3. Identified ions in UVPD-MS analysis of singly phosphorylated peaks from ABL1
    treated 3xWT, 3xS7E Mid and Sp, and 3xS2E Mid substrates (Fig. 3). The phosphorylated Tyr1
    sites were shown in blue boxes.

    8
    9
    10
    Table S4. Identified ions in UVPD-MS analysis of singly phosphorylated peaks from ABL1
    treated, GluC digested 26xS7E Sp CTD substrate (Fig. 3B and 3E-F). The phosphorylated Tyr1
    sites were shown in blue boxes.

    11
    12
    Table S5. Identified ions in UVPD-MS analysis of singly phosphorylated peaks from ABL1
    treated 3xS7R Sp, Arg1810, and Cit1810 substrates (Fig. 5A and 5C/D). The phosphorylated
    Tyr1 sites were shown in blue boxes. Tables are split across pages due to size constraints.

    13
    14
    15
    Table S6. Identified ions in UVPD-MS analysis of singly phosphorylated peaks from ABL1
    treated 3xS7K Sp and 3xS7Q Mid/Sp substrates (Fig. 6A and 6C/D). The phosphorylated Tyr1
    sites were shown in blue boxes. Tables are split across pages due to size constraints.

    16
    17
    18

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