ABSTRACT: Ribosome assembly, which requires the temporal and spatial coordination of multiple interconnected processes, is essential for life in every known organism. The large ribosomal subunit (LSU) of Escherichia coli (E. coli) contains two RNA molecules, 23S and 5S, which serve as a platform for assembly. Ribosomal RNA (rRNA) modifications and processing are key steps in this process. We simultaneously detect and quantify eight classes of 23S rRNA modifications in the mature 50S LSU from E. coli cells expressing wild-type DbpA, the 50S LSU from cells expressing the helicase-inactive R331A DbpA construct, and two LSU intermediates, 35S and 45S, which accumulate in distinct ribosome maturation pathways in R331A DbpA-expressing cells. Additionally, we analyze the 3′-end processing of 23S and 5S rRNA in all these particles. Most 23S rRNA modifications are incorporated into the 35S and 45S intermediates at levels comparable to those in the 50S LSU from both wild-type and R331A DbpA-expressing cells, indicating that modification enzymes complete their functions before 35S and 45S accumulation. However, three enzymes exhibit differential modification patterns. RlmN incorporates N²-methyladenosine (m²A) at position 2507 more extensively in the 50S LSU from wild-type DbpA-expressing cells than in the 35S, 45S, and 50S LSU from R331A DbpA-expressing cells. Conversely, RluC incorporates pseudouridine (Ψ) at position 2508 at lower levels in the 50S LSU from wild-type DbpA-expressing cells than in the 35S, 45S, and 50S LSU from R331A DbpA-expressing cells. The RluF enzyme incorporates Ψ at position 2508 at a lower level in the 50S LSU from R331A DbpA-expressing cells than in the 35S and 45S intermediates and the 50S LSU from wild-type DbpA-expressing cells. Since the pathways leading to 35S and 45S account for only ~50% of total ribosome assembly in R331A DbpA-expressing cells, these findings reveal that RluF activity is reduced in a subset of maturation pathways. The 3′ end of 23S rRNA is less mature in the early-stage LSU intermediate, 35S, and more mature in the late-stage LSU intermediate, 45S. Interestingly, the 3′ end of 5S rRNA is less processed in the 45S than in the 35S, suggesting that 5S rRNA in the 45S intermediate matures during the final steps of large subunit ribosome assembly. A complete understanding of ribosome assembly demands a precise characterization of ribosome assembly pathways and processes across diverse cellular conditions. This study reveals critical insights into rRNA modifications and processing dynamics in bacterial ribosomes under stress.
