6.5.1.3 D120A alanine-scanning mutagenesis 652387 6.5.1.3 D135A about 5% of the ligation activity of wild-type enzyme, mutant enzyme displays 4% of the adenylyltransferase activity observed with wild-type enzyme 662241 6.5.1.3 D244A site-directed mutagenesis 652429 6.5.1.3 D292A no ligation activity 662241 6.5.1.3 D292E about 10% of the ligation activity of wild-type enzyme 662241 6.5.1.3 D308A about 70% of the ligation activity of wild-type enzyme 662241 6.5.1.3 D425N lethal mutation 676215 6.5.1.3 D65A inactive 748777 6.5.1.3 D726A lethal mutation 676215 6.5.1.3 D95A the mutant shows RNA 3'-P guanylylation activity 748777 6.5.1.3 DELTA1-105 reacts with ATP to form covalent protein-adenylate adducts,mutant protein retains RNA sealing activity 662304 6.5.1.3 DELTA1-125 reacts with ATP to form covalent protein-adenylate adducts, mutant protein retains RNA sealing activity 662304 6.5.1.3 DELTA1-135 does not reacts with ATP to form covalent protein-adenylate adducts, mutant enzyme is unable to seal RNA strands 662304 6.5.1.3 E139A about as active as wild-type enzyme in RNA ligation, mutant displays near wild-type adenylyltransferase activity 662241 6.5.1.3 E151A inactive -, 748784 6.5.1.3 E185A site-directed mutagenesis 652429 6.5.1.3 E204A substitution mutation introduced into the ORFb y PCR with two-stage overlap extension method 653679 6.5.1.3 E206A the mutant shows strongly reduced activity compared to the wild type enzyme 748777 6.5.1.3 E218A lethal mutation 676215 6.5.1.3 E227A site-directed mutagenesis 652429 6.5.1.3 E227D site-directed mutagenesis 652429 6.5.1.3 E227Q site-directed mutagenesis 652429 6.5.1.3 E230A mutant is defective in phosphodiester formation at a preadenylylated nick 676230 6.5.1.3 E230A mutant is dysfunctional in ligase adenylylation 676230 6.5.1.3 E230D mutation reduces nick sealing activity to 1% of the wild-type level 676230 6.5.1.3 E230Q mutation reduces nick sealing activity to less than 1% of the wild-type level 676230 6.5.1.3 E231A inactive 748784 6.5.1.3 E256A the mutation shows a moderate effects on step 1 adenylylation (5-20fold decrease in affinity for ATP) as compared to the wild type enzyme -, 748784 6.5.1.3 E278A mutant enzyme retains adenylyltransferase and RNA ligase activities 662304 6.5.1.3 E295A about as active as wild-type enzyme in RNA ligation 662241 6.5.1.3 E296A about 10% of the ligation activity of wild-type enzyme 662241 6.5.1.3 E299A about 80% of the ligation activity of wild-type enzyme 662241 6.5.1.3 E29A about 90% of the RNA ligation activity of wild-type enzyme, mutant displays near wild-type adenylyltransferase activity 662241 6.5.1.3 E300A about 50% of the ligation activity of wild-type enzyme 662241 6.5.1.3 E305A mutant is defective in phosphodiester formation at a preadenylylated nick 676230 6.5.1.3 E305A mutant is dysfunctional in ligase adenylylation 676230 6.5.1.3 E305D mutation reduces nick sealing activity to 1% of the wild-type level 676230 6.5.1.3 E305Q mutation reduces nick sealing activity to 7% of the wild-type level 676230 6.5.1.3 E326A lethal mutation 676215 6.5.1.3 E34A alanine-scanning mutagenesis 652387 6.5.1.3 E34D substitution mutant, introduced into the rnl2 gene by PCR 652387 6.5.1.3 E34Q substitution mutant, introduced into the rnl2 gene by PCR 652387 6.5.1.3 E63A about 80% of the RNA ligation activity of wild-type enzyme, mutant displays near wild-type adenylyltransferase activity 662241 6.5.1.3 E741A lethal mutation 676215 6.5.1.3 E81A site-directed mutagenesis 652429 6.5.1.3 E95A the mutation shows a moderate effects on step 1 adenylylation (5-20fold decrease in affinity for ATP) as compared to the wild type enzyme 748784 6.5.1.3 E96A the mutation shows a moderate effects on step 1 adenylylation (5-20fold decrease in affinity for ATP) as compared to the wild type enzyme -, 748784 6.5.1.3 E99A alanine-scanning mutagenesis 652387 6.5.1.3 F119A alanine-scanning mutagenesis 652387 6.5.1.3 F175A the mutant forms traced amounts of ligase-AMP intermediates, with more than 40fold decrease in affinity for ATP 748784 6.5.1.3 F204A inactive 748777 6.5.1.3 F281A mutant is defective in phosphodiester formation at a preadenylylated nick 676230 6.5.1.3 F281A mutant is dysfunctional in ligase adenylylation 676230 6.5.1.3 F281L mutation reduces nick sealing activity to 5% of the wild-type level 676230 6.5.1.3 F76A site-directed mutagenesis 652429 6.5.1.3 F77A site-directed mutagenesis 652429 6.5.1.3 F77L site-directed mutagenesis 652429 6.5.1.3 G102A site-directed mutagenesis 652429 6.5.1.3 G168A mutant is defective in phosphodiester formation at a preadenylylated nick 676230 6.5.1.3 G168A mutant is dysfunctional in ligase adenylylation 676230 6.5.1.3 G228A site-directed mutagenesis 652429 6.5.1.3 G55A site-directed mutagenesis 652429 6.5.1.3 H1060A lethal mutation 676215 6.5.1.3 H167A mutant is dysfunctional in ligase adenylylation 676230 6.5.1.3 H167N mutation reduces nick sealing activity to 7% of the wild-type level 676230 6.5.1.3 H167Q mutation reduces nick sealing activity to less than 1% of the wild-type level 676230 6.5.1.3 H203A inactive 748777 6.5.1.3 H250A pRNA circularization by the mutant enzyme is 6% of the wild-type rate 677101 6.5.1.3 H37A substitution mutation introduced into the ORFb y PCR with two-stage overlap extension method 653679 6.5.1.3 H37D substitution mutation introduced into the ORFb y PCR with two-stage overlap extension method 653679 6.5.1.3 H404A inactive 748777 6.5.1.3 H515A lethal mutation 676215 6.5.1.3 H515N lethal mutation 676215 6.5.1.3 H515Q lethal mutation 676215 6.5.1.3 H777N lethal mutation 676215 6.5.1.3 H777Q lethal mutation 676215 6.5.1.3 K107A about as active as wild-type enzyme in RNA ligation, mutant displays near wild-type adenylyltransferase activity 662241 6.5.1.3 K109R site-directed mutagenesis 650982 6.5.1.3 K117A mutation is lethal in vivo 677101 6.5.1.3 K119A site-directed mutagenesis 652429 6.5.1.3 K119Q site-directed mutagenesis 652429 6.5.1.3 K119R site-directed mutagenesis 652429 6.5.1.3 K152A lethal mutation 676215 6.5.1.3 K165A mutant is inert in enzyme-adenylate formation and nick-joining 662304 6.5.1.3 K170A inactive 749277 6.5.1.3 K170H inactive 749277 6.5.1.3 K170M inactive 749277 6.5.1.3 K170Q inactive 749277 6.5.1.3 K184A inactive 716756 6.5.1.3 K186A mutant is dysfunctional in ligase adenylylation 676230 6.5.1.3 K186Q mutation reduces nick sealing activity to 1% of the wild-type level 676230 6.5.1.3 K186R mutation reduces nick sealing activity to 3% of the wild-type level 676230 6.5.1.3 K189A alanine-scanning mutagenesis 652387 6.5.1.3 K209A alanine-scanning mutagenesis 652387 6.5.1.3 K225A substitution mutation introduced into the ORFb y PCR with two-stage overlap extension method 653679 6.5.1.3 K227A substitution mutation introduced into the ORFb y PCR with two-stage overlap extension method 653679 6.5.1.3 K240A site-directed mutagenesis 652429 6.5.1.3 K240Q site-directed mutagenesis 652429 6.5.1.3 K240R site-directed mutagenesis 652429 6.5.1.3 K242A site-directed mutagenesis 652429 6.5.1.3 K246A inactive 748784 6.5.1.3 K273A about as active as wild-type enzyme in RNA ligation 662241 6.5.1.3 K314A about as active as wild-type enzyme in RNA ligation 662241 6.5.1.3 K315A about 90% of the ligation activity of wild-type enzyme 662241 6.5.1.3 K319A about as active as wild-type enzyme in RNA ligation 662241 6.5.1.3 K326Q mutation reduces nick sealing activity to less than 1% of the wild-type level 676230 6.5.1.3 K326R mutation reduces nick sealing activity to 3% of the wild-type level 676230 6.5.1.3 K35A substitution mutation introduced into the ORFb y PCR with two-stage overlap extension method 653679 6.5.1.3 K480A the mutant shows 2fold increased activity compared to the wild type enzyme 748777 6.5.1.3 K541A lethal mutation 676215 6.5.1.3 K543A lethal mutation 676215 6.5.1.3 K54A about 15% of the RNA ligation activity of wild-type enzyme, mutant displays near wild-type adenylyltransferase activity 662241 6.5.1.3 K73A the mutant has ATP affinity comparable to the wild type enzyme 748784 6.5.1.3 K75A site-directed mutagenesis 652429 6.5.1.3 K75Q site-directed mutagenesis 652429 6.5.1.3 K75R site-directed mutagenesis 652429 6.5.1.3 K86R site-directed mutagenesis 650982 6.5.1.3 K97A inactive -, 748784 6.5.1.3 K99A site-directed mutagenesis 652429 6.5.1.3 L104A L104A strain is temperature sensitive, no growth at 37°C 677101 6.5.1.3 L104A pRNA circularization by the mutant enzyme is 6% of the wild-type rate 677101 6.5.1.3 additional information expression of N-terminal amino acids 1-253 gives a protein defective in overall ligation but retaining the ability to form EpA intermediate, bind to pRNA and transfer AMP to pRNA, albeit less efficiently than wild-type ligase. Expression of amino acids 255-381 results in a protein that fails to form a detectable protein-RNA complex and does not support overall ligation or RNA circularization 694452 6.5.1.3 additional information S121A, S124A, and H250 strains grow at all temperatures 677101 6.5.1.3 N102A inactive 748784 6.5.1.3 N184A site-directed mutagenesis 652429 6.5.1.3 N202A inactive 748777 6.5.1.3 N309A about 70% of the ligation activity of wild-type enzyme 662241 6.5.1.3 N40A alanine-scanning mutagenesis 652387 6.5.1.3 N40D substitution mutant, introduced into the rnl2 gene by PCR 652387 6.5.1.3 N40Q substitution mutant, introduced into the rnl2 gene by PCR 652387 6.5.1.3 N40R substitution mutant, introduced into the rnl2 gene by PCR 652387 6.5.1.3 N78A site-directed mutagenesis 652429 6.5.1.3 N99A the mutation shows a moderate effects on step 1 adenylylation (5-20fold decrease in affinity for ATP) as compared to the wild type enzyme 748784 6.5.1.3 Q106A about 20% of the RNA ligation activity of wild-type enzyme, mutant displays near wild-type adenylyltransferase activity 662241 6.5.1.3 R104A the mutant forms traced amounts of ligase-AMP intermediates, with more than 40fold decrease in affinity for ATP 748784 6.5.1.3 R118A the mutant forms traced amounts of ligase-AMP intermediates, with more than 40fold decrease in affinity for ATP 748784 6.5.1.3 R155A about 35% of the ligation activity of wild-type enzyme, mutant enzyme displays 16% of the adenylyltransferase activity observed with wild-type enzyme 662241 6.5.1.3 R166A site-directed mutagenesis 652429 6.5.1.3 R182A site-directed mutagenesis 652429 6.5.1.3 R221A about 60% of the ligation activity of wild-type enzyme, mutant displays near wild-type adenylyltransferase activity 662241 6.5.1.3 R266A no ligation activity 662241 6.5.1.3 R266K about 50% of the ligation activity of wild-type enzyme 662241 6.5.1.3 R266Q no ligation activity 662241 6.5.1.3 R275A the mutation shows a moderate effects on step 1 adenylylation (5-20fold decrease in affinity for ATP) as compared to the wild type enzyme 748784 6.5.1.3 R278A the mutation shows a moderate effects on step 1 adenylylation (5-20fold decrease in affinity for ATP) as compared to the wild type enzyme -, 748784 6.5.1.3 R33A no ligation activity, mutant enzyme displays 3% of the adenylyltransferase activity observed with wild-type enzyme 662241 6.5.1.3 R463A lethal mutation 676215 6.5.1.3 R463K lethal mutation 676215 6.5.1.3 R463Q lethal mutation 676215 6.5.1.3 R511K lethal mutation 676215 6.5.1.3 R511Q lethal mutation 676215 6.5.1.3 R54A site-directed mutagenesis 652429 6.5.1.3 R54K site-directed mutagenesis 652429 6.5.1.3 R54Q site-directed mutagenesis 652429 6.5.1.3 R55A alanine-scanning mutagenesis 652387 6.5.1.3 R71A site-directed mutagenesis 652429 6.5.1.3 R76A the mutation does not have a major effect on all three steps in the ligation reaction 748784 6.5.1.3 S103A pRNA circularization by the mutant enzyme is 5% of the wild-type rate 677101 6.5.1.3 S103A S103A strain is temperature sensitive, no growth at 37°C 677101 6.5.1.3 S118A S118 strain is temperature sensitive, no growth at 37°C 677101 6.5.1.3 S170A about 5% of the ligation activity of wild-type enzyme, mutant enzyme displays 8% of the adenylyltransferase activity observed with wild-type enzyme 662241 6.5.1.3 S185N mutation reduces nick sealing activity to less than 1% of the wild-type level 676230 6.5.1.3 S185T mutation reduces nick sealing activity to 12% of the wild-type level 676230 6.5.1.3 S272A about 50% of the ligation activity of wild-type enzyme 662241 6.5.1.3 S292N about 25% of the ligation activity of wild-type enzyme 662241 6.5.1.3 S701A lethal mutation 676215 6.5.1.3 T1001A lethal mutation 676215 6.5.1.3 T117A the mutant has ATP affinity comparable to the wild type enzyme. The mutation favors the reverse RNA adenylylation reaction to deadenylate the 5'-AMP from the RNA-adenylate, thereby inhibiting phosphodiester bond synthesis 748784 6.5.1.3 T163A mutant is dysfunctional in ligase adenylylation 676230 6.5.1.3 T163S mutation reduces nick sealing activity to 2% of the wild-type level 676230 6.5.1.3 T163V mutation reduces nick sealing activity to 30% of the wild-type level 676230 6.5.1.3 T675A lethal mutation 676215 6.5.1.3 T675V lethal mutation 676215 6.5.1.3 W329A about as active as wild-type enzyme in RNA ligation 662241 6.5.1.3 Y136A about 95% of the ligation activity of wild-type enzyme, mutant displays near wild-type adenylyltransferase activity 662241 6.5.1.3 Y5A about 10% of the RNA ligation activity of wild-type enzyme, mutant enzyme displays 39% of the adenylyltransferase activity observed with wild-type enzyme 662241