C-terminal Extension of Truncated Recombinant Proteins in Escherichia coli with a 10Sa RNA Decapeptide

Abstract

When murine interleukin-6 is overexpressed in Escherichia coli, a small population of molecules exhibits a novel C-terminal modification. Peptide mapping, electrospray ionization-mass spectrometry, and automated N- and C-terminal sequencing identified a peptide ("tag" peptide), -Ala-Ala-Asn-Asp-Glu-Asn-Tyr-Ala-Leu-Ala-Ala-COOH, encoded by a small metabolically stable RNA of E. coli (10Sa RNA) attached to truncated C termini of the recombinant protein. A mutant strain of E. coli in which the chromosomal 10Sa RNA gene (ssrA) is disrupted does not produce this C-terminal modification, confirming that the tag peptide originates from the ssrA gene.

Identification of Endogenous SsrA-tagged Proteins Reveals Tagging at Positions Corresponding to Stop Codons

“…The slyD::kan disruption was constructed by inserting a kanamycin resistance cassette into the gene at a position near codon 140. 4 It appears, therefore, that tagging occurs near the site of insertion. Although tagging can be viewed as an artifact in this instance, it also serves as a reminder of a potentially important function for SsrA.…”

Proline Residues at the C Terminus of Nascent Chains Induce SsrA Tagging during Translation Termination

“…SsrA has been shown to act as both a transfer RNA and a messenger RNA (1)(2)(3)(4). SsrA has a domain that is very similar to a portion of tRNA Ala and is aminoacylated with alanine at its 3Ј end.…”

“…SsrA has a domain that is very similar to a portion of tRNA Ala and is aminoacylated with alanine at its 3Ј end. SsrA also contains a short open reading frame, which in Escherichia coli encodes the sequence ANDENYALAA (3). The tmRNA model of SsrA activity postulates that SsrA enters the empty A-site of stalled ribosomes and adds its charged alanine to the nascent polypeptide by transpeptidation.…”

Various Effects of Paromomycin on tmRNA-directed trans-Translation

“…The mRNA domain, encoding the last 10 amino acids of the 11-amino acid tag peptide, is surrounded by four pseudoknot structures in the middle of this molecule. The tag peptide was first found at the C terminus of a fraction of mouse interleukin-6 expressed in Escherichia coli (12). Later, it was also found on other polypeptides when they are translated from artificial mRNAs lacking a termination codon (4) or possessing a cluster of rare codons (13) and from endogenous mRNAs in E. coli and Bacillus subtilis (14 -16).…”