Systematic Quantification of Sequence and Structural Determinants Controlling mRNA stability in Bacterial Operons
Daniel P. Cetnar, Howard M. Salis
Abstract
, systematically varying RNase binding site characteristics, translation initiation rates, and transcriptional terminator efficiencies in the 5' untranslated region (UTR), intergenic, and 3' UTR regions, followed by measuring their mRNA levels using reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays during exponential growth. We show that introducing long single-stranded RNA into 5' UTRs reduced mRNA levels by up to 9.4-fold and that lowering translation rates reduced mRNA levels by up to 11.8-fold. We also found that RNase binding sites in intergenic regions had much lower effects on mRNA levels. Surprisingly, changing the transcriptional termination efficiency or introducing long single-stranded RNA into 3' UTRs had no effect on upstream mRNA levels. From these measurements, we developed and validated biophysical models of ribosome protection and RNase activity with excellent quantitative agreement. We also formulated design rules to rationally control a mRNA's stability, facilitating the automated design of engineered genetic systems with desired functionalities.