4.5S RNA, the product of the essential ffs gene of Escherichia coli, is a highly conserved, 114-base molecule that functions as a component of the signal recognition particle (SRP) and also has been implicated in protein synthesis. To address conflicting results reported in other studies and determine the essential function of 4.5S RNA, we have developed an improved genetic system to characterize ffs mutants. Multiple ffs alleles altered at several highly conserved positions were constructed. To test the ability of the alleles to complement an ffs knockout mutation in single copy, a method to transfer the genes to F' factors with minimal manipulation was developed. These F' factors along with plasmids that express ffs in multiple copy number were used to assess the ability of mutant 4.5S RNA to function in vivo. Despite the high degree of evolutionary conservation, only a single base (C62) was indispensable for RNA function under all conditions tested. To determine the importance of these bases for the interaction between 4.5S RNA and either Ffh or EF-G, an assay to measure the interaction of Ffh or EF-G with mutant 4.5S RNA molecules in vivo was also developed. Defects in Ffh binding correlated with loss of SRP-dependent protein localization and loss of viability. Defects in EF-G binding were found to correlate with a slight decrease in protein synthesis but did not correlate with loss of viability. These results clarify inconsistencies from prior studies and further support that 4.5S RNA is essential only as a component of the SRP. These results also yielded convenient methods to study the function of multiple alleles and to qualitatively measure an interaction between a protein and an RNA.