High resolution view of bacteriophage lambda gene expression by ribosome profiling
Bacteriophage lambda is one of the most extensively studied organisms, and has been a primary model for understanding basic modes of genetic regulation.  Here we examine the progress of lambda gene expression during phage development by ribosome profiling, and thereby provide a very high resolution view of lambda gene expression.  The known genes are expressed in a predictable fashion, authenticating the analysis.  But many previously unappreciated potential open reading frames become apparent in the expression analysis, revealing an unexpected complexity in the pattern of lambda gene function. Overall design: We chose temperature induction of the classic cI857 repressor mutation in a lysogen of E. coli MG1655 in order to synchronize the lytic process, sampling the lysogen and control non-lysogen both before and 2, 5, 10, and 20 minutes after shifting the temperature from 32° to 42°.  The last sample time was chosen to be before any significant cell lysis, but during the later stages of lytic gene expression. Total protected nucleotides within open reading frames were summed to determine the density of translation of each reading frame.  We take this number to indicate the overall rate of translation, although obviously this assumes that pauses in translation do not excessively affect the overall rate.  Since the expression level is not normalized for the copy number of the replicating phage DNA, it thus encompasses both the effect of DNA template availability on mRNA synthesis and the efficiency of utilization of messengers by ribosomes.