Abstract
In bacteria, mRNA decay is a major mechanism for regulating gene expression. In Escherichia coli, mRNA decay initiates with endonucleolytic cleavage by RNase E. Translating ribosomes impede RNase E cleavage, thus providing stability to mRNA. In transcripts containing multiple cistrons, the translation of each cistron initiates separately. The effect of internal translation initiations on the decay of polycistronic transcripts remains unknown, which we have investigated here using the four-cistron galETKM transcript. We find that RNase E cleaves a few nucleotides (14–36) upstream of the translation initiation site of each cistron, generating decay intermediates galTKM, galKM, and galM mRNA with fewer but full cistrons. Blocking translation initiation reduced stability, particularly of the mutated cistrons and when they were the 5′-most cistrons. This indicates that, together with translation failure, the location of the cistron is important for its elimination. The instability of the 5′-most cistron did not propagate to the downstream cistrons, possibly due to translation initiation there. Cistron elimination from the 5′ end was not always sequential, indicating that RNase E can also directly access a ribosome-free internal cistron. The finding in gal operon of mRNA decay by cistron elimination appears common in E. coli and Salmonella.
Highlights
In bacteria, mRNA concentration is modulated to cope with a rapidly changing environment
We performed northern blot of total RNA prepared from wildtype E. coli cells (MG1655) grown in Luria broth (LB) supplemented with 0.5% galactose to OD600 of 0.6
We detected five mRNA species with sizes of 4.3, 3.3, 2.3, 1.3, and 1.1 kb. We named these mRNA species galETKM, galTKM, galKM, galM1, and galM, respectively, as we show later that these mRNA species have their 5 ends at the beginning of each cistron but have the same 3 end, which is at the end of the operon (Figure 1A)
Summary
MRNA concentration is modulated to cope with a rapidly changing environment. RNase E can target mRNAs for the initial cleavage either by first sensing a 5 monophosphate group (5 end-dependent pathway) or by bypassing this requirement (direct access pathway) (Mackie, 2013; Hui et al, 2014). The N-terminal sensor domain of RNase E allows it to bind preferentially to the mono-phosphorylated rather than to the tri- or di-phosphorylated 5 end of mRNA (Mackie, 1998; Jiang and Belasco, 2004; Callaghan et al, 2005). The 5 end-dependent access requires prior conversion of transcripts with 5 tri- or di-phosphorylated end Polycistronic mRNA Decay in E. coli to mono-phosphorylated end by RNA pyrophosphohydrolase (RppH) (Celesnik et al, 2007; Deana et al, 2008). The 3 portion mRNA could be subjected to decay by further rounds of cleavage by RNase E by either of the pathways (Joyce and Dreyfus, 1998; Spickler et al, 2001; Hui et al, 2014)
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