The alternative sigma factor RpoS is a central regulator of the stress response in many Proteobacteria, acting both during exponential growth and in stationary phase. The small protein Crl increases the interaction between RpoS and RNA polymerase and thereby activates certain RpoS-dependent promoters. However, the growth-phase dependence of the interaction of Crl with different forms of polymerase remains unknown. We use 41 GFP transcriptional fusions to study the dynamics of gene regulation by RpoS and Crl during growth transition from exponential to stationary phase in Escherichia coli. We confirm that RpoS can regulate gene expression in exponential phase, both positively and negatively. Crl slightly stimulates transcription by RpoS in exponential phase and controls a subset of RpoS-dependent genes in stationary phase. Growth temperature strongly affects induction of specific promoters by RpoS, whereas its impact on gene regulation by Crl is much less significant. In addition, we identify five new genes regulated by Crl (ada, cbpA, glgS, sodC and flgM) and demonstrate that Crl improves promoter binding and opening by RpoS-containing RNA polymerase at the hdeA promoter. Our study also shows that Crl is a cognate enhancer of RpoS activity under different growth conditions, since its deletion has no effect on genes transcribed by other sigma factors.