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Ribosome-controlled transcription termination is essential for the production of antibiotic microcin C.

Zukher I, Novikova M, Tikhonov A, Nesterchuk MV, Osterman IA, Djordjevic M, Sergiev PV, Sharma CM, Severinov K - Nucleic Acids Res. (2014)

Bottom Line: Ribosome binding also makes the mccA RNA exceptionally stable.Together, these two effects-ribosome-induced transcription termination and stabilization of the message-account for very high abundance of the mccA transcript that is essential for McC production.The general scheme appears to be evolutionary conserved as ribosome-induced transcription termination also occurs in a homologous operon from Helicobacter pylori.

View Article: PubMed Central - PubMed

Affiliation: Institute of Gene Biology of the Russian Academy of Sciences, Moscow, Russia Waksman Institute for Microbiology and Department of Molecular Biology and Biochemistry, Rutgers, the State University of New Jersey, Piscataway, NJ, USA St. Petersburg State Polytechnical University, St. Petersburg, Russia.

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Ribosomes stimulate transcription termination in the mccA–mccB intergenic region in vitro. (A) Stalled E. coli RNA polymerase transcription elongation complexes containing a 34-nt long radioactively labeled RNA were formed on a DNA template containing the T7 A1 promoter fused to a fragment of mcc operon extending from position +1 to position +154. Transcription elongation was allowed to resume by the addition of NTPs (lane 1), or NTPs with PURExpress ΔRibosome (lane 2), Δ(aa, tRNA) (lane 3) or full system (lane 4). An autoradiograph of a denaturing gel showing reaction product separation is shown. (B) In vitro transcribed mccA RNA was combined with PURExpress ΔRibosome (lane 2), Δ(aa, tRNA) (lane 3) or full system (lanes 4 and 5) at conditions identical to those used in panel A experiment, and toeprinting was performed. In lane 5, reaction contained 50 μM thiostrepton. The products of toeprinting reactions were resolved by denaturing gel-electrophoresis and revealed by autoradiography. The 3′ end of the cDNA products synthesized in the toeprint experiment and labeled +17/20 correspond to bound ribosomes with the first (+17) or second (+20) codons of mccA located in the ribosomal P site (27). (C) Transcription complexes were formed as described in A on wild-type or mutant transcription templates with mutated first codon of the mccA gene (ATG1->TAA) or substitutions in the mccA SD sequence and transcription elongation was allowed to proceed. (D) In vitro transcribed wild-type mccA RNA or RNAs harboring mccA (ATG1->TAA) or SD sequence substitutions were subjected to toeprinting reaction in the presence or in the absence of PURExpress Δ(aa, tRNA) system. All designations are as in panel B.
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Figure 4: Ribosomes stimulate transcription termination in the mccA–mccB intergenic region in vitro. (A) Stalled E. coli RNA polymerase transcription elongation complexes containing a 34-nt long radioactively labeled RNA were formed on a DNA template containing the T7 A1 promoter fused to a fragment of mcc operon extending from position +1 to position +154. Transcription elongation was allowed to resume by the addition of NTPs (lane 1), or NTPs with PURExpress ΔRibosome (lane 2), Δ(aa, tRNA) (lane 3) or full system (lane 4). An autoradiograph of a denaturing gel showing reaction product separation is shown. (B) In vitro transcribed mccA RNA was combined with PURExpress ΔRibosome (lane 2), Δ(aa, tRNA) (lane 3) or full system (lanes 4 and 5) at conditions identical to those used in panel A experiment, and toeprinting was performed. In lane 5, reaction contained 50 μM thiostrepton. The products of toeprinting reactions were resolved by denaturing gel-electrophoresis and revealed by autoradiography. The 3′ end of the cDNA products synthesized in the toeprint experiment and labeled +17/20 correspond to bound ribosomes with the first (+17) or second (+20) codons of mccA located in the ribosomal P site (27). (C) Transcription complexes were formed as described in A on wild-type or mutant transcription templates with mutated first codon of the mccA gene (ATG1->TAA) or substitutions in the mccA SD sequence and transcription elongation was allowed to proceed. (D) In vitro transcribed wild-type mccA RNA or RNAs harboring mccA (ATG1->TAA) or SD sequence substitutions were subjected to toeprinting reaction in the presence or in the absence of PURExpress Δ(aa, tRNA) system. All designations are as in panel B.

Mentions: While our inability to detect the mccA transcript in cells where ribosome binding to mccA was disrupted made it impossible to determine stabilities of mutant transcripts, the result hinted that the free short transcript is unstable or, more interestingly, that ribosome binding may somehow stimulate short transcript production. To address this issue, in vitro transcription and transcription-translation experiments were performed. Since mcc promoter has very low basal transcription activity in vitro (data not shown), we used a chimeric transcription template containing a strong T7 A1 promoter with modified initial transcribed sequence (extending from positions −67 to +30, 5′-tccagatcccgaaaatttatcaaaaagagtattgacttaaagtctaacctataggatacttacagccAtcgagagggccacggcgaacagcaaccca-3′, the promoter consensus elements are underlined, the +1 position is capitalized) fused to a fragment of mcc DNA from position +1 to +154 with respect to the mcc promoter transcription start point. Transcription templates containing mutations at the mccA initiating codon and/or its SD sequence (above) were also created. Transcription templates were combined with E. coli RNA polymerase σ70 holoenzyme and transcription was initiated with a C−1pA+1pU+2pC+3 primer, ATP, GTP and radioactive CTP. Since the first adenine in the template strand is located in position +35 of the template, transcription is stalled before this position. The stalled transcription complexes contain a radioactive nascent 34-nt long RNA whose extension can be monitored upon the addition of UTP. As can be seen from Figure 4A, lane 2, very little, if any, transcription termination was detected with the wild-type template. However, when elongation of the nascent 34-mer RNA was performed in the presence of defined PURExpress in vitro translation system, a new transcript whose 3′ end matched the 3′ end of mccA RNA observed in vivo was produced on the wild-type template (Figure 4A, lane 5). The PURExpress system used in Figure 4A, lane 5, contains all components of the translation machinery and ribosomes. To determine whether transcription termination in the mccA–mccB intergenic region requires translation of the mccA gene, in vitro transcription of wild-type template was performed in the presence of PURExpress (Δaa, tRNA) system lacking amino acids and tRNA (Figure 4A, lane 4), or in the presence of PURExpress ΔRibosome system that lacks ribosomes but contains all other translation system components (Figure 4A, lane 3). As can be seen, transcription termination required the presence of ribosomes but was not dependent on the presence of amino acids and tRNA.


Ribosome-controlled transcription termination is essential for the production of antibiotic microcin C.

Zukher I, Novikova M, Tikhonov A, Nesterchuk MV, Osterman IA, Djordjevic M, Sergiev PV, Sharma CM, Severinov K - Nucleic Acids Res. (2014)

Ribosomes stimulate transcription termination in the mccA–mccB intergenic region in vitro. (A) Stalled E. coli RNA polymerase transcription elongation complexes containing a 34-nt long radioactively labeled RNA were formed on a DNA template containing the T7 A1 promoter fused to a fragment of mcc operon extending from position +1 to position +154. Transcription elongation was allowed to resume by the addition of NTPs (lane 1), or NTPs with PURExpress ΔRibosome (lane 2), Δ(aa, tRNA) (lane 3) or full system (lane 4). An autoradiograph of a denaturing gel showing reaction product separation is shown. (B) In vitro transcribed mccA RNA was combined with PURExpress ΔRibosome (lane 2), Δ(aa, tRNA) (lane 3) or full system (lanes 4 and 5) at conditions identical to those used in panel A experiment, and toeprinting was performed. In lane 5, reaction contained 50 μM thiostrepton. The products of toeprinting reactions were resolved by denaturing gel-electrophoresis and revealed by autoradiography. The 3′ end of the cDNA products synthesized in the toeprint experiment and labeled +17/20 correspond to bound ribosomes with the first (+17) or second (+20) codons of mccA located in the ribosomal P site (27). (C) Transcription complexes were formed as described in A on wild-type or mutant transcription templates with mutated first codon of the mccA gene (ATG1->TAA) or substitutions in the mccA SD sequence and transcription elongation was allowed to proceed. (D) In vitro transcribed wild-type mccA RNA or RNAs harboring mccA (ATG1->TAA) or SD sequence substitutions were subjected to toeprinting reaction in the presence or in the absence of PURExpress Δ(aa, tRNA) system. All designations are as in panel B.
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Figure 4: Ribosomes stimulate transcription termination in the mccA–mccB intergenic region in vitro. (A) Stalled E. coli RNA polymerase transcription elongation complexes containing a 34-nt long radioactively labeled RNA were formed on a DNA template containing the T7 A1 promoter fused to a fragment of mcc operon extending from position +1 to position +154. Transcription elongation was allowed to resume by the addition of NTPs (lane 1), or NTPs with PURExpress ΔRibosome (lane 2), Δ(aa, tRNA) (lane 3) or full system (lane 4). An autoradiograph of a denaturing gel showing reaction product separation is shown. (B) In vitro transcribed mccA RNA was combined with PURExpress ΔRibosome (lane 2), Δ(aa, tRNA) (lane 3) or full system (lanes 4 and 5) at conditions identical to those used in panel A experiment, and toeprinting was performed. In lane 5, reaction contained 50 μM thiostrepton. The products of toeprinting reactions were resolved by denaturing gel-electrophoresis and revealed by autoradiography. The 3′ end of the cDNA products synthesized in the toeprint experiment and labeled +17/20 correspond to bound ribosomes with the first (+17) or second (+20) codons of mccA located in the ribosomal P site (27). (C) Transcription complexes were formed as described in A on wild-type or mutant transcription templates with mutated first codon of the mccA gene (ATG1->TAA) or substitutions in the mccA SD sequence and transcription elongation was allowed to proceed. (D) In vitro transcribed wild-type mccA RNA or RNAs harboring mccA (ATG1->TAA) or SD sequence substitutions were subjected to toeprinting reaction in the presence or in the absence of PURExpress Δ(aa, tRNA) system. All designations are as in panel B.
Mentions: While our inability to detect the mccA transcript in cells where ribosome binding to mccA was disrupted made it impossible to determine stabilities of mutant transcripts, the result hinted that the free short transcript is unstable or, more interestingly, that ribosome binding may somehow stimulate short transcript production. To address this issue, in vitro transcription and transcription-translation experiments were performed. Since mcc promoter has very low basal transcription activity in vitro (data not shown), we used a chimeric transcription template containing a strong T7 A1 promoter with modified initial transcribed sequence (extending from positions −67 to +30, 5′-tccagatcccgaaaatttatcaaaaagagtattgacttaaagtctaacctataggatacttacagccAtcgagagggccacggcgaacagcaaccca-3′, the promoter consensus elements are underlined, the +1 position is capitalized) fused to a fragment of mcc DNA from position +1 to +154 with respect to the mcc promoter transcription start point. Transcription templates containing mutations at the mccA initiating codon and/or its SD sequence (above) were also created. Transcription templates were combined with E. coli RNA polymerase σ70 holoenzyme and transcription was initiated with a C−1pA+1pU+2pC+3 primer, ATP, GTP and radioactive CTP. Since the first adenine in the template strand is located in position +35 of the template, transcription is stalled before this position. The stalled transcription complexes contain a radioactive nascent 34-nt long RNA whose extension can be monitored upon the addition of UTP. As can be seen from Figure 4A, lane 2, very little, if any, transcription termination was detected with the wild-type template. However, when elongation of the nascent 34-mer RNA was performed in the presence of defined PURExpress in vitro translation system, a new transcript whose 3′ end matched the 3′ end of mccA RNA observed in vivo was produced on the wild-type template (Figure 4A, lane 5). The PURExpress system used in Figure 4A, lane 5, contains all components of the translation machinery and ribosomes. To determine whether transcription termination in the mccA–mccB intergenic region requires translation of the mccA gene, in vitro transcription of wild-type template was performed in the presence of PURExpress (Δaa, tRNA) system lacking amino acids and tRNA (Figure 4A, lane 4), or in the presence of PURExpress ΔRibosome system that lacks ribosomes but contains all other translation system components (Figure 4A, lane 3). As can be seen, transcription termination required the presence of ribosomes but was not dependent on the presence of amino acids and tRNA.

Bottom Line: Ribosome binding also makes the mccA RNA exceptionally stable.Together, these two effects-ribosome-induced transcription termination and stabilization of the message-account for very high abundance of the mccA transcript that is essential for McC production.The general scheme appears to be evolutionary conserved as ribosome-induced transcription termination also occurs in a homologous operon from Helicobacter pylori.

View Article: PubMed Central - PubMed

Affiliation: Institute of Gene Biology of the Russian Academy of Sciences, Moscow, Russia Waksman Institute for Microbiology and Department of Molecular Biology and Biochemistry, Rutgers, the State University of New Jersey, Piscataway, NJ, USA St. Petersburg State Polytechnical University, St. Petersburg, Russia.

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Related in: MedlinePlus