L4 and L22, proteins of the large ribosomal subunit, contain globular surface domains and elongated “tentacles” that reach into the core of the large subunit to form part of the lining of the peptide exit tunnel. Mutations in the L4 and L22 tentacles confer macrolide resistance to a variety of bacteria. In Escherichia coli ysine to Glutamine mutation at codon 63 of L4 and a deletion removing Methionine–Lysine–Arginine corresponding to codons 82 through 84 in L22 have been reported. Through the analysis of novel L4 and L22 mutants previously isolated in our lab, the work described here not only extends our knowledge of the influence of mutations in L4 and L22, but also provides key insights into ribosomal protein and ribosomal RNA interactions during ribosome assembly and function. Ten novel L4 and L22 mutants were characterized for their effects on bacterial physiology, translation rate, ribosome assembly, ribosomal RNA processing, erythromycin binding to ribosomes, and ribosomal RNA structure. All mutants exhibited slower growth rates compared to the parent and all had reduced in vivo s of peptide chain elongation. Large insertions in L4 and L22 resulted in very slow growth and accumulation of abnormal ribosomal subunits. All mutants also showed increased levels of precursor 23S rRNA. The canonical L4 mutant binds erythromycin poorly, whereas the canonical L22 mutant binds erythromycin well. The L4 mutation was thus proposed to narrow the exit tunnel while the L22 mutation was proposed to widen the tunnel. However, this explanation is not supported as a general model since drug binding studies show that not all of the novel L4 mutants bind erythromycin poorly and not all of the newly identified L22 mutants bind erythomycin well. Biochemical structure probing analysis of 23S rRNA revealed changes in several nucleotides of the peptidyl transferase associated region (PTAR) of 23S rRNA in some of the L4 and L22 mutants. Particularly, nucleotides at the catalytic center of the ribosome showed altered conformation in the L4 mutants with the slowest elongation rates. These results highlight the important roles of L4 and L22 in ribosome assembly, structure and function.