SsrA-mediated tagging and proteolysis of LacI and its role in the regulation of lac operon

Abstract

SsrA RNA of Escherichia coli, also known as 10Sa RNA or tmRNA, acts both as tRNA and mRNA when ribosomes are paused at the 3' end of an mRNA lacking a stop codon. This process, referred to as trans-translation, leads to the addition of a short peptide tag to the C-terminus of the incomplete nascent polypeptide. The tagged polypeptide is then degraded by C-terminal-specific proteases. Here, we focused on endogenous targets for the SsrA system and on a potential regulatory role of SsrA RNA. First, we show that trans-translation events occur frequently in normally growing E. COLI: cells. More specifically, we report that the lacI mRNA encoding Lac repressor (LacI) is a specific natural target for trans-translation. The binding of LacI to the lac operators results in truncated lacI mRNAs that are, in turn, recognized by the SsrA system. The SsrA-mediated tagging and proteolysis of LacI appears to play a role in cellular adaptation to lactose availability by supporting a rapid induction of lac operon expression.

Fig. 1. Expression of artificially tagged CRPs in wild-type and ssrA cells. (A) Structure of CRP-AA and CRP-DD. Amino acid sequences of the C-terminal portion of CRP derivatives are shown along with corresponding nucleotide sequences. Asterisks indicate the stop codon. The MluI site (5′-ACGCGT-3′) used to introduce DNA fragments encoding the AA- or DD-tag is underlined. (B) Effect of the ssrA mutation on the expression of CRP, CRP-AA and CRP-DD proteins. Total proteins equivalent to OD₆₀₀ = 0.01, prepared from PP47 (lanes 1, 3 and 5) and PP47ΔssrA (lanes 2, 4 and 6) cells harboring pCRP (lanes 1 and 2), pCRP-AA (lanes 3 and 4) or pCRP-DD (lanes 5 and 6), were analyzed by western blotting using anti-CRP antibody. (C) Northern blot analysis of the crp mRNAs. Total RNAs (2.5 µg), prepared from PP47 (lanes 1 and 3) and PP47ΔssrA (lanes 2 and 4) cells harboring pCRP-AA (lanes 1 and 2) or pCRP-DD (lanes 3 and 4), were subjected to northern blot analysis using a fluorescein-labeled probe for crp mRNAs.

Fig. 2. (A) Schematic drawing of alanyl-SsrA^AA and alanyl-SsrA^DD RNAs. The nucleotides mutated in the tag-coding region of SsrA^DD are underlined. (B) Detection of tagging of cellular proteins. Total proteins equivalent to OD₆₀₀ = 0.1, prepared from PP47ΔssrA cells harboring pSTV28 (lane 1), pSsr-AA (lane 2) and pSsr-DD (lane 3), were analyzed by western blotting using anti-DD-tag antibody. Lane 4 represents total protein equivalent to OD₆₀₀ = 0.01, prepared from PP47ΔssrA cells harboring pCRP-DD. The arrow indicates the artificially tagged CRP-DD.

Fig. 3. (A) Schematic drawing of the E.coli lacPO region. O1 and O3 are shown as open boxes. O3 partially overlaps the lacI ORF. Closed boxes indicate the parts of lacI and lacZ ORFs. The arrow indicates the transcription start site of lacP. (B) Nucleotide sequences of the wild-type and modified lac operators. Mutations introduced into the lacPO region are indicated by lowercase (O1^– and O3^–) or underlined letters (O3^id). The last two amino acid residues of LacI, GQ, were changed to EL in the O3^id mutant. Asterisks indicate the lacI stop codon.

Fig. 4. Western blot analysis of the LacI protein produced in cells harboring pIT613. MC4100ΔssrA cells harboring pIT613 were transformed with pSTV28 (lanes 2 and 7), pSsr-AA (lanes 3 and 6) or pSsr-DD (lanes 4 and 5). Total proteins equivalent to OD₆₀₀ = 0.01 were analyzed by western blotting using anti-LacI antibody (lanes 1–4) or anti-DD-tag antibody (lanes 5–8). The position of intact LacI is indicated by an asterisk. The dashed arrows indicate major truncated LacI proteins recognized by anti-LacI antibody in ΔssrA cells. The arrow indicates an elongated tagged LacI recognized by anti-LacI antibody. The major tagged LacI proteins recognized by anti-DD-tag antibody are indicated by a bracket. Lanes 1 and 8 represent total proteins equivalent to OD₆₀₀ = 0.01 prepared from MC4100ΔssrA cells harboring pBR322/pSTV28 and pBR322/pSsr-DD, respectively.

Fig. 5. Effect of IPTG on LacI tagging. MC4100ΔssrA cells harboring both pIT613 and pSsr-DD were grown in the presence (lanes 2 and 4) or the absence (lanes 1 and 3) of 1 mM IPTG. Total proteins equivalent to OD₆₀₀ = 0.01 were analyzed by western blotting using anti-LacI or anti-DD-tag antibodies. The position of intact LacI is indicated by asterisks. The arrow indicates an elongated tagged LacI recognized by anti-LacI antibody. The major tagged LacI proteins recognized by anti-DD-tag antibody are indicated by a bracket.

Fig. 6. Effect of operator mutations on LacI tagging. The pIT613 derivatives containing the lacO variants indicated were introduced into MC4100ΔssrA cells harboring pSsr-DD. The nucleotide sequences of lacO variants are shown in Figure 3. Cells were grown in the presence or the absence of 1 mM IPTG and total proteins equivalent to OD₆₀₀ = 0.01 were analyzed by western blotting using anti-DD-tag antibody. The position of intact LacI is indicated by an asterisk. The major tagged LacI proteins recognized by anti-DD-tag antibody are indicated by a bracket.

Fig. 7. Model for trans-translation acting on the lacI mRNA. Formation of the O1–LacI–O3 repression DNA loop may cause premature termination of transcription resulting in truncated lacI mRNAs that would be recognized by the SsrA system.

Fig. 8. LacI tagging in the wild-type lacI gene. MC4100ΔssrA harboring pIT455 was transformed with vector plasmid pSTV28 (lane 2), pSsr-AA (lane 3) or pSsr-DD (lanes 4 and 5). MC4100ΔssrA harboring both pBR322 and pSTV28 was also subjected to the analysis as a control (lane 1). Cells were grown in the presence (lane 5) or the absence (lanes 1–4) of 1 mM IPTG and total proteins equivalent to OD₆₀₀ = 0.2 were analyzed by western blotting using anti-LacI antibody. The position of intact LacI is indicated by an asterisk. The arrow indicates an extended tagged LacI recognized by anti-LacI antibody.

Fig. 9. Effect of the ssrA mutation on lac operon expression. (A) Expression of the lac operon in response to the addition of IPTG. W3110 (squares) and its ssrA derivative (circles) were grown in LB medium. IPTG was added to the culture at OD₆₀₀ = 0.3 to a final concentration of 0.1 mM and the β-galactosidase activity at various time points was measured. (B) Expression of the lac operon in W3110 (upper panel) and its ssrA derivative (lower panel) in response to the consumption of glucose in the medium. The strains were grown in M9 minimal medium containing 0.04% glucose and 0.2% lactose as carbon sources. Optical density at 600 nm (squares) and β-galactosidase activity (diamonds) of the samples were determined at the time points indicated.