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The heat shock response (HSR) is an essential cellular and organismal protective mechanism against acute forms of physiological and environmental stress.In E.coli,the heat shock response to temperature upshift from 32 to 42℃ consists in induction of synthesis of more than 20 heat shock proteins and various molecular chaperones.However,there has not been a comprehensive genetic analysis of the HSR regulatory pathway,in part because traditional forward genetic screens are inadequately suited to identify genes that regulate the HSR.Here,we adapted ribosome footprinting to chloramphenicol-treated cells to generate a genome-wide translational profiling in E.coli under the condition of heat shock,and identified more than 600 previously unidentified open reading frames,which accounted for about 15% of known genes.Meanwhile,we used LC-MS to verify whether the newly identified genes expressed and explored the relationship between these new genes and the HSR.