Characterization of 16S rRNA Processing with Pre-30S Subunit Assembly Intermediates from E. coli

Area of Science:

• Molecular Biology
• Microbiology
• Genetics

Background:

• Ribosomal RNA (rRNA) is essential for protein synthesis.
• 16S rRNA, a key component of the bacterial small ribosomal subunit, is crucial for mRNA decoding.
• The maturation of precursor rRNA (pre-rRNA) involves precise nucleolytic processing, with 3'-end maturation being less understood than 5'-end maturation.

Purpose of the Study:

• To investigate the molecular mechanisms and enzymes involved in the 3'-end maturation of bacterial 16S rRNA.
• To identify the specific nucleolytic activities responsible for processing precursor 16S rRNA (17S rRNA) within small subunit (SSU) assembly intermediates.

Main Methods:

• Utilized purified in vivo-formed small subunit (SSU) assembly intermediates (pre-SSUs) containing 17S rRNA from wild-type Escherichia coli.
• Incubated pre-SSUs with E. coli S100 cell extracts and purified candidate enzymes (RNase R, RNase II, PNPase, RNase PH, YbeY).
• Analyzed the processing of 17S rRNA into mature 16S rRNA and intermediate products using in vitro assays.

Main Results:

• Exonucleases RNase R, RNase II, PNPase, and RNase PH demonstrated nucleolytic activity on the 3'-end of pre-SSU 17S rRNA in vitro.
• The endonuclease YbeY did not show nucleolytic activity on pre-SSUs under the tested conditions.
• Identified dominant maturation pathways: either complete 5'-end processing before 3'-end maturation, or concurrent 5' and 3' end processing.

Conclusions:

• Bacterial 16S rRNA maturation is a multifaceted process involving multiple redundant enzymatic pathways.
• Escherichia coli possesses complementary pathways for SSU biogenesis, allowing it to compensate for the inactivation of single enzymes.
• The identified exonucleases play significant roles in the 3'-end maturation of 16S rRNA, contributing to the robustness of ribosomal biogenesis.