Leaky ribosomal scanning enables tunable translation of bicistronic ORFs in green algae
成果类型：
Article
署名作者：
Duenas, Marco A.; Crai, Rory J.; Gallaher, Sean; Mosele, Jeffre L.; Merchant, Sabeeha S.
署名单位：
University of California System; University of California Berkeley; University of California System; University of California Berkeley; University of California System; University of California Berkeley; United States Department of Energy (DOE); Lawrence Berkeley National Laboratory
刊物名称：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
ISSN/ISSBN：
0027-14322
DOI：
10.1073/pnas.2417695122
发表日期：
2025-03-04
关键词：
messenger-rna
initiation
expression
SEQUENCES
mechanism
upstream
reinitiation
transcript
codons
MODEL
摘要：
Advances in sequencing technology have unveiled examples of nucleus- encoded polycistrons, once considered rare. Exclusively polycistronic transcripts are prevalent in green algae, although the mechanism by which multiple polypeptides are translated from a single transcript is unknown. Here, we used bioinformatic and in vivo mutational analyses to evaluate competing mechanistic models for translation of bicistronic mRNAs in green algae. High- confidence manually curated datasets of bicistronic loci from two revealed a preference for weak Kozak- like sequences for ORF 1 and an underrepresentation of potential initiation codons before the ORF 2 start codon, which are suitable conditions for leaky ribosome scanning to allow ORF 2 translation. We used mutational analysis in A. protothecoides to test the mechanism. In vivo manipulation of the ORF weaker Kozak- like sequence enhancing expression and a stronger one diminishing it. A synthetic bicistronic dual reporter demonstrated inversely adjustable activity of green fluorescent protein expressed from ORF 1 and luciferase from ORF 2, depending on the of multiple ORFs in green algal bicistronic transcripts is consistent with episodic leaky yotic genomes and for transgene expression in synthetic biology applications.