Erythromycin Resistance Peptides Selected from Random Peptide Libraries

Abstract

It was assumed for a long time that the ribosome is indifferent to the sequence of the polypeptide it is synthesizing. New evidence, however, indicates that nascent or newly synthesized polypeptides can affect functions of the ribosome in cis. In a number of cases, the newly translated peptide exerts its effect on translation while still being located within the ribosome. For example, short nascent peptides regulate stalling of the ribosome on mRNA, which is required for inducing the expression of chloramphenicol resistance (cat and cmlA) and erythromycin resistance (erm) genes (1, 2). Ribosome stalling depends on the amino acid sequence of the nascent peptide rather than on the nucleotide sequence of mRNA and occurs when the nascent peptide is only several amino acids long and should be located within the ribosome. Other examples include: translational bypass of the coding gap in bacteriophage T4 gene 60 mRNA (ribosome "hopping"), which depends on the amino acid sequence of the nascent peptide (3); dependence of termination efficiency on the amino acid sequence of the nascent peptide (4); attenuation of eukaryotic gene expression by short upstream open reading frames, which depends on the encoded amino acid sequences but not the mRNA sequence; and others (5-7). Despite a growing number of cases where cis-action of the newly synthesized peptide on the ribosome has been either demonstrated or suspected, almost nothing is known about molecular mechanisms of interaction between the ribosome and regulatory cis-acting peptides. The nature and location of the peptideresponsive site remains obscure.A new example of a cis-acting peptide is represented by a pentapeptide encoded in Escherichia coli 23 S rRNA (6). It was demonstrated that production in E. coli cells of a 34-nucleotidelong segment of 23 S rRNA, positions 1235-1268 (8), renders cells resistant to the ribosome-targeted antibiotic erythromycin (8, 9). Curiously, erythromycin resistance was mediated by translation of a pentapeptide (E-peptide) 1 encoded in the rRNA fragment. Mutations that affected translation initiation signals of the E-peptide mini-gene (Shine-Dalgarno region and initiator GUG codon) abolished erythromycin resistance. Interestingly, mutations at the terminator UAA codon, as well as some missense mutations, also interfered with peptide activity, suggesting that the size of the peptide and its amino acid sequence are essential for its functions. Translation of the E-peptide mRNA in the cell-free system rendered ribosomes resistant to erythromycin. However, addition of the synthetic E-peptide to the translating ribosome in vitro did not confer any erythromycin resistance (8). Thus, it appears that the E-peptide acts in cis so that only the ribosome on which the peptide has been translated becomes resistant to the drug. The single binding site of erythromycin is located on the large ribosomal subunit in the vicinity of the peptidyltransferase center. Accordingly, one possible mechanism of the E-peptide action is that the newly translated peptide ...

Macrolide resistance mechanisms inEnterobacteriaceae: Focus on azithromycin

“…Moreover, the same study showed that alterations in the size of the peptide possess a strong effect on the resistance levels conferred, suggesting that the Leu residue plays a key role in the dissociation of erythromycin. This finding may be in accordance with the high consensus of previously studied E-peptides selected using erythromycin, which usually presents at least one Leu at position 3 (Tenson et al, 1997). However, this E-peptide model of action cannot be extended to all macrolides.…”

“…Further studies using random peptide libraries have shown a variety of synthetic short peptides of between three and six amino acids able to produce macrolide resistance as well as the inability of these peptides to affect the MIC of erythromycin when included in longer amino acid sequences (Tenson et al, 1997). However, the Shine-Dalgarno region is located in a hairpin at position 1198-1247, being difficult to access by RNA-polymerases (Dam et al, 1996) and probably being unexpressed in normal conditions.…”

A novel site of antibiotic action in the ribosome: Interaction of evernimicin with the large ribosomal subunit

“…24. A mRNA transcript with the sequence GGGAAUUCGGGCCCUU-GUUAACAAUUAAGGAGGUAUACUAUGAAUGCAAUA-AAUAACUGCAG(A) 21 (25) was prepared by in vitro transcription and purified according to ref. 26.…”

A Bright Future for Antibiotics?

“…Importantly, macrolide binding seems to be involved in cellular processes. Thus, stalling by specific nascent peptides, a cellular mechanism used for regulation of expression of several bacterial and eukaryotic genes, is sensitive to signals connected with macrolide binding (73)(74)(75)(76)(77)(78)(79)(80)(81)(82)(83).…”