Roles of the 5'-phosphate sensor domain in RNase E

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• RNase E is a key bacterial enzyme regulating RNA processing and degradation.
• The 5 eal-phosphate sensor of RNase E plays a role in its autoregulation and substrate specificity.
• Deletions in the C-terminal domain of RNase E affect its function and stability.

Purpose of the Study:

• To investigate the functional consequences of mutations in the RNase E 5 eal-phosphate sensor, particularly when combined with deletions in its C-terminal domain.
• To elucidate the role of the 5 eal-phosphate sensor in RNase E autoregulation and substrate targeting.
• To understand the mechanisms of substrate recognition and RNA processing hierarchy mediated by RNase E.

Main Methods:

• Site-directed mutagenesis was used to introduce specific mutations (R169Q, T170A) into the RNase E 5 eal-phosphate sensor.
• Deletions were introduced in the non-catalytic C-terminal domain of RNase E.
• Phenotypic analysis was performed to assess lethality, RNase E expression levels, proteolytic fragment accumulation, and the stability of specific RNA targets (rpsT P1 mRNA, 16S rRNA).
• The processing of other model mRNAs and tRNA precursors was also examined.

Main Results:

• Combinations of 5 eal-phosphate sensor mutations and C-terminal deletions resulted in lethality.
• Mutations in the phosphate sensor led to strong overexpression of RNase E and accumulation of proteolytic fragments, indicating impaired autoregulation.
• Stabilization of rpsT P1 mRNA (up to sixfold) and slowed maturation of 16S rRNA were observed with phosphate sensor mutations.
• The decay of other model mRNAs and processing of tRNA precursors remained unaffected by these mutations.

Conclusions:

• The 5 eal-phosphate sensor is crucial for efficient RNase E autoregulation and proper maturation of specific RNA species like 16S rRNA.
• Overlapping mechanisms of substrate recognition exist for RNase E, establishing a hierarchy for RNA target processing.
• The interplay between the 5 eal-phosphate sensor and the C-terminal domain is essential for RNase E viability and function.