Using DNA sequencing electrophoresis compression artifacts as reporters of stable mRNA structures affecting gene expression.
The formation of secondary structure in oligonucleotide DNA is known to lead to "compression" artifacts in electropherograms produced through DNA sequencing. Separately, the formation of secondary structure in mRNA is known to suppress translation; in particular, when such structures form in a region covered by the ribosome either during, or shortly after, initiation of translation. Here, we demonstrate how a DNA sequencing compression artifact provides important clues to the location(s) of translation-suppressing secondary structural elements in mRNA. Our study involves an engineered version of a gene sourced from Rhodothermus marinus encoding an enzyme called Cel12A. We introduced this gene into Escherichia coli with the intention of overexpressing it, but found that it expressed extremely poorly. Intriguingly, the gene displayed a remarkable compression artifact during DNA sequencing electrophoresis. Selected "designer" silent mutations destroyed the artifact. They also simultaneously greatly enhanced the expression of the cel12A gene, presumably by destroying stable mRNA structures that otherwise suppress translation. We propose that this method of finding problem mRNA sequences is superior to software-based analyses, especially if combined with low-temperature CE.
Kapoor
Divya
D
Division of Protein Science and Engineering, Institute of Microbial Technology, Chandigarh, India.
Chandrayan
Sanjeev Kumar
SK
Ahmed
Shubbir
S
Guptasarma
Purnananda
P
D016477:Artifacts; D001426:Bacterial Proteins; D001483:Base Sequence; D004269:DNA, Bacterial / Q000032:analysis; D019075:Electrophoresis, Capillary / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis / Q000235:genetics*; D020871:RNA Stability / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D045245:Rhodothermus / Q000201:enzymology / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012333:RNA, Messenger