Bidirectional regulation of translational reading-frame switch by ribosome-flanking mRNA duplexes

Autor: Wan-Ping Huang, 黃婉蘋
Rok vydání: 2019
Druh dokumentu: 學位論文 ; thesis
Popis: 107
When a translating ribosome encounters a signal of programmed ribosomal frameshifting (PRF) in an mRNA, it can switch into an alternative reading-frame, and synthesizes distinct protein products. It has been known that a slippery sequence X_XXY_YYZ and a downstream duplex mRNA structure with an appropriate spacer length stimulates the reading-frame to switch one nucleotide backward to the 5’end of mRNA (-1 PRF). By contrast, an insufficient decoding in ribosomal A site can cause the reading-frame to shift one nucleotide forward to the 3’ end of an mRNA (+1 PRF). In E. coli, dnaX and prfB utilize different frameshifting signals to promote -1 PRF and +1 PRF, respectively. dnaX contains a slippery sequence A_AAA_AAG and a downstream mRNA hairpin to stimulate -1 PRF, and produce γ subunit of DNA polymerase III. By contrast, prfB encodes mature release factor 2 (RF2) via +1 PRF at the slippery sequence CUU_UGA when RF2 is insufficient. Besides, an internal Shine-Dalgarno (SD) sequence adjacent to the ribosomal E site upstream of their slippery sequence can pair with the 3’ end sequence of 16S rRNA to a form a duplex, and acts as a stimulator of +1 PRF, or an attenuator of -1 PRF. Because a refolding mRNA hairpin in the upstream of a slippery sequence in eukaryotes has regulation activity similar to the SD-ASD duplex of prfB and dnaX, it raises the question that if an upstream mRNA hairpin can replace the SD-ASD duplexes to regulate +1 PRF and -1 PRF in prokaryotes. In a cell-free system of E. coli, a stable mRNA hairpin upstream of A_AAA_AAG slippery site is shown to attenuate -1 PRF, which is stimulated by a downstream pseudoknot. In contrast, it stimulates +1 PRF in CUU_UGA site in the absence of RF2. These results suggest that the regulatory activity of a refolding mRNA hairpin is conserved in both eukaryotic and prokaryotic translation process. Furthermore, a pseudoknot downstream of CUU_UGA site can attenuate +1 PRF, whereas stimulate -1 PRF in the absence of RF2. Mass and mutagenesis analysis suggest that the non-canonical -1 PRF occurs after tRNA translocation, or before next aminoacyl-tRNA accommodation with the ribosomal A site remaining empty as long as there is a NNX_XNN_UGA frameshift site (X_X represents two identical nucleotides). Additionally, the mRNA duplexes flanking the frameshift site CUU_UGA can counteract the regulation activities of each other. Similar results were also observed when UGA was replaced by a hungry codon. Finally, selected endogenous genes in E. coli genome with potential non-canonical -1 PRF signal (NNX_XXN_UGA) were identified. The potential -1 PRF signals derived from four candidate genes were cloned into split-superfolder GFP reporter for further -1 PRF analysis.
Databáze: Networked Digital Library of Theses & Dissertations
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