Escherichia coli DinB (DNA polymerase IV) possesses an enzyme architecture resulting in specialized lesion bypass function and the potential for creating -1 frameshifts in homopolymeric nucleotide runs. We have previously shown that the mutagenic potential of DinB is regulated by the DNA damage response protein UmuD(2). In the current study, we employ a pre-steady-state fluorescence approach to gain a mechanistic understanding of DinB regulation by UmuD(2). Our results suggest that DinB, like its mammalian and archaeal orthologs, uses a template slippage mechanism to create single base deletions on homopolymeric runs. With 2-aminopurine as a fluorescent reporter in the DNA substrate, the template slippage reaction results in a prechemistry fluorescence change that is inhibited by UmuD(2). We propose a model in which DNA templates containing homopolymeric nucleotide runs, when bound to DinB, are in an equilibrium between non-slipped and slipped conformations. UmuD(2), when bound to DinB, displaces the equilibrium in favor of the non-slipped conformation, thereby preventing frameshifting and potentially enhancing DinB activity on non-slipped substrates.