Escherichia coli (E. coli) ribosomal protein (r-protein) L4 has extra-ribosomal biological functions. Previously, we described L4 as inhibiting RNase E activity through protein-protein interactions. Here, we report that from stabilized transcripts regulated by L4-RNase E, mRNA levels of tnaA (encoding tryptophanase from the tnaCAB operon) increased upon ectopic L4 expression, whereas TnaA protein levels decreased. However, at non-permissive temperatures (to inactivate RNase E), tnaA mRNA and protein levels both increased in an rnets mutant. Thus, L4 protein fine-tunes TnaA protein levels independently of its inhibition of RNase E. We demonstrate that ectopically-expressed L4 binds with transcribed spacer RNA between tnaC and tnaA and down-regulates TnaA translation. We found that deletion of the 5′- or 3′-half of the spacer compared to the wild type resulted in a similar reduction in TnaA translation in the presence of L4. In vitro binding of L4 in tnaC-tnaA transcribed spacer RNA results in changes to its secondary structure. We reveal that during early stationary phase bacterial growth, steady-state levels of tnaA mRNA increased, but TnaA protein levels decreased. We further confirm that endogenous L4 binds to tnaC-tnaA transcribed spacer RNA in cells at early stationary phase. Our results reveal the novel function of L4 in fine-tuning TnaA protein levels during cell growth and demonstrate that r-protein L4 acts as a translation regulator outside the ribosome and its own operon.