RNA Structural Modulation in the Heart of the Ribosome

Abstract

DEAD-box RNA helicase DbpA is one of the RNA maturation factors that E. coli employs during its ribosome assembly process. DbpA binds tightly and specifically to hairpin 92 of the 23S ribosomal RNA which is located in the peptidyl transferase center. Therefore, DbpA is implicated in RNA structural rearrangement in a ribosome region that is crucial for cell survival. When the helicase inactive R331A DbpA construct is expressed in E. coli cells, a 45S particle accumulates. This particle is a misassembled intermediate of the large ribosome subunit. It is not known if the 45S misassembled particle rearranges inside the cell and forms the active 50S large ribosome subunit, or if the resulting RNA structural misfolding is so severe that the 45S particle is designated by the cell for degradation. To understand the fate of the 45S particle in the cell, the ability of the 45S particle to form a native 50S subunit is tested by pulse chase. First, in the cell expressing R331A DbpA and lacking the wild type DbpA from their genome, RNA is labeled with [5,6-3H] uridine for a specific amount of time, and then transcription of new RNA is stopped by the addition of rifampicin. Cell culture aliquots are obtained at a series of time points after stopping the transcription of new RNA, and ribosomal profile analyses are performed using sucrose gradient ultracentrifugation. The ribosome profile experiments demonstrate that the conversion of the 45S intermediate to the 50S large subunit particle does occur in the cells. The conversion rate of the 45S particle to the 50S particle is currently being measured.