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The 16s ribosomal RNA decoding domain in translational initiation and the interaction between 23s ribosomal RNA fragment and protein L1: A mutational analysis
Abstract
The 3$\sp\prime$ minor domain of the 16S rRNA is associated with ribosomal decoding. Host cells expressing C1395U or C1395U/G1505U mutant rRNAs are inviable or slow-growing, respectively; those accumulating G1505U mutant rRNA show growth similar to wild-type. At the third doubling after expression of the mutant rRNA, host cells containing C1395U or C1395U/G1505U accumulated larger amounts of free 50S and 30S subunits than those containing the wild-type or G1505U mutant rRNA. The proportion of mutant rRNA reached 50% in 30S subunit populations but was marginal in 70S subunits and polysomes in all three mutant-containing strains. 30S subunits containing C1395U or C1395U/G1505U rRNA exhibited a marginal tRNA binding capacity and no response to IF2 stimulation while those containing mutant G1505U rRNA possessed an affinity for tRNA similar to their wild-type counterparts but failed to respond to IF2. These observations imply that mutations in decoding domain of 16S rRNA provoke changes related to translational initiation, and that the accumulation of the mutant 30S subunits impedes the function of the wild-type 30S subunits in the host cell. The binding site for ribosomal protein Ll lies within a sequence spanning nucleotides 2067-2235 of the E. coli 23S rRNA. A DNA fragment encoding the Ll-specific rRNA was inserted into a T3 transcription vector and subjected to random and site-directed mutagenesis. Wild-type and mutant transcripts were then prepared, and their affinities for Ll were determined by a quantitative filter assay. The results were as follows. (1) Mutations that disrupted base pairing within helix 77 significantly reduced the affinity of Ll for the rRNA. (2) The replacement of conserved purines at positions 2126 and 2168-2173 in the loop between helices 77 and 78 also severely impaired Ll-rRNA interaction. (3) Deletion of 1-4 non-conserved bases from the same loop, however, led to less than a 50% reduction in Ll association. Nondenaturing polyacrylamide gel electrophoresis revealed that most of the mutant rRNA transcripts exhibiting defects in Ll binding also migrated more slowly than their wild-type counterparts, indicating that they were improperly folded. Conformational differences in several of these transcripts were pinpointed through an analysis of their susceptibility to chemical modification. Finally, a smaller RNA transcript, encompassing helices 77 and 78 and loop 77/78, was synthesized and found to be active in binding protein Ll.
Subject Area
Molecular biology|Microbiology
Recommended Citation
Dong, Peining, "The 16s ribosomal RNA decoding domain in translational initiation and the interaction between 23s ribosomal RNA fragment and protein L1: A mutational analysis" (1994). Doctoral Dissertations Available from Proquest. AAI9420620.
https://scholarworks.umass.edu/dissertations/AAI9420620