The signal recognition particle (SRP) is a ribonucleoprotein complex whose components are highly conserved throughout all three domains of life, where it functions to target proteins to extracytoplasmic locations. In Escherichia coli, the SRP is comprised of a single essential protein (Ffh) in complex with a 4.5S RNA species. To better understand how the structure of Ffh contributes to its function, we have used genetic approaches to isolate and characterize new ffh mutants altered in two distinct domains of the protein. Both domains of interest have been implicated as being important for binding to hydrophobic signal peptides of membrane proteins. These studies include using a random sequence approach to identify amino acids important for activity of the finger loop domain. The finger loop was identified from structural analysis as a ∼ 20 amino acid domain with the unusual properties of being both hydrophobic and exposed near the surface of Ffh. Approximately 1% of the random sequences were able to replace the FL domain of Ffh. Bioinformatic analysis of the random sequences revealed that all of the complementing sequences followed a trend of high hydrophobicity at the amino-terminus that decreased towards the carboxy end. These observations were validated by observing that mutants that deviated from this trend rendered Ffh nonfunctional. Mutants were characterized by growth rates that allowed the sequences to be grouped into three functional classes. Secondary and tertiary structure predictions suggested that the products of the random sequences lack extensive secondary structure, which is consistent with the role of the finger loop in binding a variety of ligands. To address the importance of the conserved methionine residues at the carboxy-terminal region (M-domain) in SRP function, we combined phylogenetic comparisons with functional studies, including replacing methionine residues within the M-domain with other residues that varied in hydrophobicity, side chain flexibility and charge. These studies revealed that, in E. coli, the M-domain of Ffh was able to tolerate substitutions of five different hydrophobic amino acids including valine, phenylalanine, tyrosine, tryptophan and isoleucine for the conserved methionine residues found in helix αM4 and the extreme C-terminus. Phylogenetic comparisons of microorganisms with varying optimal growth temperatures revealed methionine residues were substituted with amino acids containing less flexible side chains. Interestingly, we observed that mutants containing less flexible residues were able to support cell viability at higher growth temperatures better than at lower temperatures. Phylogenetic comparisons also revealed three positions where methionine is highly conserved. We show that replacing all of the methionine residues, except these three highly conserved residues, with valine yielded a functional SRP. In contrast to predicted results, these studies reveal that the M-domain of Ffh is highly flexible in content and that methionines are not absolutely required for SRP function. Collectively, these efforts have contributed to our understanding of SRP function by identifying key features essential for the function of the signal sequence binding domain of the Ffh protein component of SRP.