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Modular domain organization of RNase E and PNPase Miao, Xin


mRNA decay in Escherichia coli is carried out and controlled by concerted actions of a number of ribonucleases and other protein factors. In order to gain insights into the catalytic mechanisms and regulation involved in this important cellular process, we have utilized mutational analysis to study two key enzymes, RNase E and PNPase. RNase E is the endoribonuclease that initiates the degradation of bulk mRNA. We constructed a series of truncated Rne proteins to delineate the domains of RNase E. Our data show that the catalytic site of RNase E is located between residues 208 and 407. The first 207 residues, encompassing an SI domain, may play a role in maintaining structural integrity of the protein. We have also identified a minimal Arg-rich RNA-binding site between residues 608 and 622. Preliminary data indicate that the RBD plays a role in facilitating degradation and processing of some RNAs. In addition, this study confirms the findings by others that RNase E displays a modular domain organization. PNPase, a highly conserved protein in bacteria and plants, is one of two.major 3'-5' exoribonucleases degrading RNA fragments generated by RNase E cleavages. Based on the available sequence information, we constructed a series of deletion mutants to express individual domains and to study their functions. The data show that the catalytic site resides within the PH domain (residues 312-541). The PH' domain (residues 1-210), related to the PH domain, is required for structural integrity. Interestingly, Rne-binding activities are detected in both N-terminal portion (PH' domain plus the linker) of the Pnp protein and the C-terminal SI domain (residues 622-690). The mode of Pnp-Rne interaction is discussed. Our biochemical evidence correlates well with the published crystal structure of PNPase.

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