| Crystal structure of Escherichia coli PNPase:Central channel residues are involved in processive RNA degradation |
| ZHONGHAO SHI,1,2 WEI-ZEN YANG,1 SUE LIN-CHAO,1 KIN-FU CHAK,2 and HANNA S. YUAN1,3 . Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China 2. Institute of Biochemistry, National Yang-Ming University, Taipei, Taiwan, Republic of China 3. Graduate Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China Bacterial
polynucleotide phosphorylase (PNPase) plays a major role in mRNA
turnover by the degradation of RNA from the 39-to 59-ends. Here, we
determined the crystal structures of the wild-type and a C-terminal
KH/S1 domain-truncated mutant (DKH/S1) of Escherichia coli PNPase at
resolutions of 2.6 A° and 2.8 A° , respectively. The six RNase PH
domains of the trimeric PNPase assemble into a ring-like structure
containing a central channel. The truncated mutant DKH/S1 bound and
cleaved RNA less efficiently with an eightfold reduced binding
affinity. Thermal melting and acid-induced trimer dissociation studies,
analyzed by circular dichroism and dynamic light scattering, further
showed that DKH/S1 formed a less stable trimer than the full-length
PNPase. The crystal structure of DKH/S1 is more expanded, containing a
slightly wider central channel than that of the wild-type PNPase,
suggesting that the KH/S1 domain helps PNPase to assemble into a more
compact trimer, and it regulates
the channel size allosterically. Moreover, site-directed mutagenesis of
several arginine residues in the channel neck regions produced
defective PNPases that either bound and cleaved RNA less efficiently or
generated longer cleaved oligonucleotide products, indicating that
these arginines were involved in RNA binding and processive
degradation. Taking these results together, we conclude that the
constricted central channel and the basic-charged residues in the
channel necks of PNPase play crucial roles in trapping RNA for
processive exonucleolytic degradation.
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