Literature summary for 2.7.7.19 extracted from

Betat, H.; Rammelt, C.; Martin, G.; Moerl, M.
Exchange of regions between bacterial poly(A) polymerase and the CCA-adding enzyme generates altered specificities (2004), Mol. Cell, 15, 389-398.

Cloned(Commentary)

Cloned (Comment)	Organism
vector pET30-EK/LIC, expression in Escherichia coli	Escherichia coli

Protein Variants

Protein Variants	Comment	Organism
additional information	chimeras of CCA-adding enzyme and PAP were constructed	Escherichia coli

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.00039	-	oligo(A)12	incubation 30 min at 30 °C	Escherichia coli
0.13	-	ATP	incubation 30 min at 30 °C	Escherichia coli
0.27	-	CTP	incubation 30 min at 30 °C	Escherichia coli

Organism

Organism	UniProt	Comment	Textmining
Escherichia coli	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
The wild-type poly(A) polymerase, as well as deletion variants are expressed in Escherichia coli BL21(DE3) (Novagen). Freshly transformed cells are grown at 28-37°C in 700 ml LB medium containing 0.030 mg/ml kanamycin and 0.033 mg/ml chloramphenicol. At mid-log phase (A600 = 0.6), expression is induced by the addition of IPTG to a final concentration of 0.200 mM. After 3-5 hr of incubation at 28-37°C, cells are harvested by centrifugation and lysed by lysozyme treatment and sonication in ice-cold buffer I (20 mM Tris/HCl pH 7.6, 0.5 M NaCl, 5 mM imidazole and 0.75 mg/ml lysozyme). After centrifugation for 30 min at 25000 xg and 4°C, the proteins in the supernatant are purified by FPLC on a 5 ml HiTrap Chelating Sepharose column (Amersham Biosciences) and eluted with 500 mM imidazole. Fractions containing the enzymes as determined by SDS-PAGE are pooled, dialyzed against buffer II (20 mM Tris/HCl pH 7.6, 0.5 M NaCl, 5 mM MgCl2 and 10% glycerol). All proteins are stored in the presence of 40% (v/v) glycerol at -20°C.	Escherichia coli

Purification (Comment)

Organism

The wild-type poly(A) polymerase, as well as deletion variants are expressed in Escherichia coli BL21(DE3) (Novagen). Freshly transformed cells are grown at 28-37°C in 700 ml LB medium containing 0.030 mg/ml kanamycin and 0.033 mg/ml chloramphenicol. At mid-log phase (A600 = 0.6), expression is induced by the addition of IPTG to a final concentration of 0.200 mM. After 3-5 hr of incubation at 28-37°C, cells are harvested by centrifugation and lysed by lysozyme treatment and sonication in ice-cold buffer I (20 mM Tris/HCl pH 7.6, 0.5 M NaCl, 5 mM imidazole and 0.75 mg/ml lysozyme). After centrifugation for 30 min at 25000 xg and 4°C, the proteins in the supernatant are purified by FPLC on a 5 ml HiTrap Chelating Sepharose column (Amersham Biosciences) and eluted with 500 mM imidazole. Fractions containing the enzymes as determined by SDS-PAGE are pooled, dialyzed against buffer II (20 mM Tris/HCl pH 7.6, 0.5 M NaCl, 5 mM MgCl2 and 10% glycerol). All proteins are stored in the presence of 40% (v/v) glycerol at -20°C.

Escherichia coli

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
ATP + oligo(A)12	-	Escherichia coli	diphosphate + oligo(A)13	-	?
ATP + RNAn	-	Escherichia coli	diphosphate + RNAn+1	-	?
CTP + RNAn	-	Escherichia coli	diphosphate + RNAn+1	-	?

Synonyms

Synonyms	Comment	Organism
PAP	-	Escherichia coli
poly(A) polymerase	-	Escherichia coli

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism	Structure
0.43	-	CTP	incubation 30 min at 30 °C	Escherichia coli