Genetic analysis of polynucleotide phosphorylase structure and functions.
Polynucleotide phosphorylase (PNPase) is a phosphate-dependent 3' to 5' exonuclease widely diffused among bacteria and eukaryotes. The enzyme, a homotrimer, can also be found associated with the endonuclease RNase E and other proteins in a heteromultimeric complex, the RNA degradosome. PNPase negatively controls its own gene (pnp) expression by destabilizing pnp mRNA. A current model of autoregulation maintains that PNPase and a short duplex at the 5'-end of pnp mRNA are the only determinants of mRNA stability. During the cold acclimation phase autoregulation is transiently relieved and cellular pnp mRNA abundance increases significantly. Although PNPase has been extensively studied and widely employed in molecular biology for about 50 years, several aspects of structure-function relationships of such a complex protein are still elusive. In this work, we performed a systematic PCR mutagenesis of discrete pnp regions and screened the mutants for diverse phenotypic traits affected by PNPase. Overall our results support previous proposals that both first and second core domains are involved in the catalysis of the phosphorolytic reaction, and that both phosphorolytic activity and RNA binding are required for autogenous regulation and growth in the cold, and give new insights on PNPase structure-function relationships by implicating the alpha-helical domain in PNPase enzymatic activity.
Briani
Federica
F
Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy. federica.briani@unimi.it
Del Favero
Marta
M
Capizzuto
Rossana
R
Consonni
Chiara
C
Zangrossi
Sandro
S
Greco
Claudio
C
De Gioia
Luca
L
Tortora
Paolo
P
Dehò
Gianni
G
D015152:Blotting, Northern; D003080:Cold Temperature; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015870:Gene Expression; D005786:Gene Expression Regulation; D009154:Mutation; D016133:Polymerase Chain Reaction; D011117:Polyribonucleotide Nucleotidyltransferase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D020871:RNA Stability / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics
D012333:RNA, Messenger; D011117:Polyribonucleotide Nucleotidyltransferase