Identifying the RNA Kinetic Traps in Ribosome Assembly

Abstract

The ribosome is the cellular machine responsible for protein production in every living cell. While the final structure of the mature ribosome is known from x-ray crystallography studies, the assembly process remains largely unknown. To fulfill the cells’ demand for proteins, ribosome assembly in vivo occurs fast and efficiently. Consequently, intermediates of the assembly process are short lived and do not accumulate in large amounts for investigation. To increase the accumulation of in vivo intermediates of large subunit ribosome assembly in Escherichia coli, we take advantage of the helicase inactive DbpA construct R331A. When R331A DbpA is expressed in cells, three large subunit particles, with sedimentation coefficients of 27S, 35S, and 45S, accumulate. The particles rearrange to form the native 50S large subunit via parallel pathways. We interrogated the RNA structure of the 45S particle using dimethyl sulfate and SHAPE chemical probing reagents combined with Next Generation Sequencing. The structural probing experiments revealed perturbed secondary structures of the P-loop and the helices of the peptidyltransferase center. Additionally, it revealed an incorrect orientation of helix 38, the finger of the A-site, and several critical, broken tertiary RNA contacts between distant regions of the 23S rRNA. The data suggests the peptidyltransferase center's maturation is one of the final events during assembly. This severe misfolding explains the slow rearrangement of the 45S particle to the native 50S subunit. Next, we will characterize the RNA structure of the 27S and 35S particles. The structural probing of the 27S and 35S particles will provide insights into the early steps of ribosome assembly, which remain a complete mystery.