0 18 Ubiquitin-mediated ubiquitin-mediated JJ 19 29 processing processing NN 30 32 of of IN 33 41 NF-kappa NF-kappa NNP 42 43 B B NNP 44 59 transcriptional transcriptional JJ 60 69 activator activator NN 70 79 precursor precursor NN 80 84 p105 p105 NN 84 85 . . . 86 100 Reconstitution Reconstitution NNP 101 103 of of IN 104 105 a a DT 106 115 cell-free cell-free JJ 116 122 system system NN 123 126 and and CC 127 141 identification identification NN 142 144 of of IN 145 148 the the DT 149 166 ubiquitin-carrier ubiquitin-carrier NN 167 174 protein protein NN 174 175 , , , 176 178 E2 E2 NNP 178 179 , , , 180 183 and and CC 184 185 a a DT 186 191 novel novel JJ 192 209 ubiquitin-protein ubiquitin-protein JJ 210 216 ligase ligase NN 216 217 , , , 218 220 E3 E3 NNP 220 221 , , , 222 230 involved involve VBN 231 233 in in IN 234 245 conjugation conjugation NN 245 246 . . . 248 250 In in IN 251 255 most most JJS 256 261 cases case NNS 261 262 , , , 263 266 the the DT 267 282 transcriptional transcriptional JJ 283 289 factor factor NN 290 298 NF-kappa NF-kappa NNP 299 300 B B NNP 301 303 is be VBZ 304 305 a a DT 306 317 heterodimer heterodimer NN 318 328 consisting consist VBG 329 331 of of IN 332 335 two two CD 336 344 subunits subunit NNS 344 345 , , , 346 349 p50 p50 NN 350 353 and and CC 354 357 p65 p65 NN 357 358 , , , 359 364 which which WDT 365 368 are be VBP 369 376 encoded encode VBN 377 379 by by IN 380 383 two two CD 384 392 distinct distinct JJ 393 398 genes gene NNS 399 401 of of IN 402 405 the the DT 406 409 Rel Rel NNP 410 417 family. family. NN 418 421 p50 p50 NN 422 424 is be VBZ 425 435 translated translate VBN 436 438 as as IN 439 440 a a DT 441 450 precursor precursor NN 451 453 of of IN 454 457 105 105 CD 458 461 kDa kda NN 461 462 . . . 463 466 The the DT 467 477 C-terminal c-terminal JJ 478 484 domain domain NN 485 487 of of IN 488 491 the the DT 492 501 precursor precursor NN 502 504 is be VBZ 505 512 rapidly rapidly RB 513 521 degraded degrade VBN 521 522 , , , 523 530 forming form VBG 531 534 the the DT 535 541 mature mature JJ 542 545 p50 p50 NN 546 553 subunit subunit NN 554 563 consisted consist VBD 564 566 of of IN 567 570 the the DT 571 581 N-terminal n-terminal JJ 582 588 region region NN 589 591 of of IN 592 595 the the DT 596 604 molecule molecule NN 604 605 . . . 606 609 The the DT 610 619 mechanism mechanism NN 620 622 of of IN 623 633 generation generation NN 634 636 of of IN 637 640 p50 p50 NN 641 643 is be VBZ 644 647 not not RB 648 653 known know VBN 653 654 . . . 655 657 It it PRP 658 661 has have VBZ 662 666 been be VBN 667 676 suggested suggest VBN 677 681 that that IN 682 685 the the DT 686 706 ubiquitin-proteasome ubiquitin-proteasome JJ 707 713 system system NN 714 716 is be VBZ 717 725 involved involve VBN 726 728 in in IN 729 732 the the DT 733 740 process process NN 740 741 ; ; : 742 749 however however RB 749 750 , , , 751 754 the the DT 755 763 specific specific JJ 764 771 enzymes enzyme NNS 772 780 involved involve VBN 781 784 and and CC 785 788 the the DT 789 798 mechanism mechanism NN 799 801 of of IN 802 809 limited limited JJ 810 821 proteolysis proteolysis NN 821 822 , , , 823 825 in in IN 826 831 which which WDT 832 836 half half NN 837 839 of of IN 840 843 the the DT 844 852 molecule molecule NN 853 855 is be VBZ 856 862 spared spar VBN 862 863 , , , 864 868 have have VBP 869 873 been be VBN 874 881 obscure obscure JJ 881 882 . . . 883 893 Palombella Palombella NNP 894 897 and and CC 898 908 colleagues colleague NNS 909 910 ( ( ( 910 920 Palombella Palombella NNP 920 921 , , , 922 926 V.J. V.J. NNP 926 927 , , , 928 933 Rando Rando NNP 933 934 , , , 935 939 O.J. O.J. NNP 939 940 , , , 941 949 Goldberg Goldberg NNP 949 950 , , , 951 955 A.L. A.L. NNP 955 956 , , , 957 960 and and CC 961 969 Maniatis Maniatis NNP 969 970 , , , 971 979 T.(1994) T.(1994) NNP 980 984 Cell Cell NNP 985 987 78 78 CD 987 988 , , , 989 996 773-785 773-785 CD 996 997 ) ) ) 998 1002 have have VBP 1003 1008 shown show VBN 1009 1013 that that IN 1014 1023 ubiquitin ubiquitin NN 1024 1026 is be VBZ 1027 1035 required require VBN 1036 1039 for for IN 1040 1043 the the DT 1044 1054 processing processing NN 1055 1057 in in IN 1058 1059 a a DT 1060 1069 cell-free cell-free JJ 1070 1076 system system NN 1077 1079 of of IN 1080 1081 a a DT 1082 1091 truncated truncate VBN 1091 1092 , , , 1093 1105 artificially artificially RB 1106 1117 constructed construct VBN 1117 1118 , , , 1119 1125 60-kDa 60-kda JJ 1126 1135 precursor precursor NN 1135 1136 . . . 1137 1141 They they PRP 1142 1146 have have VBP 1147 1151 also also RB 1152 1157 shown show VBN 1158 1162 that that IN 1163 1173 proteasome proteasome NN 1174 1184 inhibitors inhibitor NNS 1185 1190 block block VBP 1191 1194 the the DT 1195 1205 processing processing NN 1206 1210 both both CC 1211 1213 in in FW 1214 1219 vitro vitro FW 1220 1223 and and CC 1224 1226 in in FW 1227 1231 vivo vivo FW 1231 1232 . . . 1233 1235 In in IN 1236 1240 this this DT 1241 1246 study study NN 1246 1247 , , , 1248 1250 we we PRP 1251 1262 demonstrate demonstrate VBP 1263 1277 reconstitution reconstitution NN 1278 1280 of of IN 1281 1282 a a DT 1283 1292 cell-free cell-free JJ 1293 1303 processing processing NN 1304 1310 system system NN 1311 1314 and and CC 1315 1326 demonstrate demonstrate VBP 1327 1335 directly directly RB 1336 1340 that that IN 1340 1341 : : : 1342 1343 ( ( ( 1343 1344 a a LS 1344 1345 ) ) ) 1346 1349 the the DT 1350 1370 ubiquitin-proteasome ubiquitin-proteasome JJ 1371 1377 system system NN 1378 1380 is be VBZ 1381 1389 involved involve VBN 1390 1392 in in IN 1393 1403 processing processing NN 1404 1406 of of IN 1407 1410 the the DT 1411 1417 intact intact JJ 1418 1422 p105 p105 NN 1423 1432 precursor precursor NN 1432 1433 , , , 1434 1435 ( ( ( 1435 1436 b b NN 1436 1437 ) ) ) 1438 1449 conjugation conjugation NN 1450 1452 of of IN 1453 1462 ubiquitin ubiquitin NN 1463 1465 to to TO 1466 1469 the the DT 1470 1479 precursor precursor NN 1480 1482 is be VBZ 1483 1485 an an DT 1486 1495 essential essential JJ 1496 1508 intermediate intermediate JJ 1509 1513 step step NN 1514 1516 in in IN 1517 1520 the the DT 1521 1531 processing processing NN 1531 1532 , , , 1533 1534 ( ( ( 1534 1535 c c NN 1535 1536 ) ) ) 1537 1540 the the DT 1541 1549 recently recently RB 1550 1560 discovered discover VBN 1561 1566 novel novel JJ 1567 1574 species specie NNS 1575 1577 of of IN 1578 1581 the the DT 1582 1599 ubiquitin-carrier ubiquitin-carrier NN 1600 1607 protein protein NN 1607 1608 , , , 1609 1614 E2-F1 E2-F1 NNP 1614 1615 , , , 1616 1620 that that WDT 1621 1623 is be VBZ 1624 1632 involved involve VBN 1633 1635 in in IN 1636 1639 the the DT 1640 1651 conjugation conjugation NN 1652 1655 and and CC 1656 1667 degradation degradation NN 1668 1670 of of IN 1671 1674 p53 p53 NN 1674 1675 , , , 1676 1678 is be VBZ 1679 1683 also also RB 1684 1692 required require VBN 1693 1696 for for IN 1697 1700 the the DT 1701 1708 limited limited JJ 1709 1719 processing processing NN 1720 1722 of of IN 1723 1726 the the DT 1727 1731 p105 p105 NN 1732 1741 precursor precursor NN 1741 1742 , , , 1743 1746 and and CC 1747 1748 ( ( ( 1748 1749 d d LS 1749 1750 ) ) ) 1751 1752 a a DT 1753 1758 novel novel JJ 1758 1759 , , , 1760 1773 approximately approximately RB 1774 1781 320-kDa 320-kda JJ 1782 1789 species specie NNS 1790 1792 of of IN 1793 1810 ubiquitin-protein ubiquitin-protein JJ 1811 1817 ligase ligase NN 1817 1818 , , , 1819 1821 is be VBZ 1822 1830 involved involve VBN 1831 1833 in in IN 1834 1837 the the DT 1838 1845 process process NN 1845 1846 . . . 1847 1851 This this DT 1852 1857 novel novel JJ 1858 1864 enzyme enzyme NN 1865 1867 is be VBZ 1868 1876 distinct distinct JJ 1877 1881 from from IN 1882 1887 E6-AP E6-AP NNP 1887 1888 , , , 1889 1892 the the DT 1893 1908 p53-conjugating p53-conjugating JJ 1909 1915 ligase ligase NN 1915 1916 , , , 1917 1920 and and CC 1921 1925 from from IN 1926 1928 E3 e3 NN 1929 1934 alpha alpha NN 1934 1935 , , , 1936 1939 the the DT 1940 1941 “ `` `` 1941 1946 N-end n-end NN 1947 1951 rule rule NN 1951 1952 ” '' '' 1953 1959 ligase ligase NN 1959 1960 . . .