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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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The mccA transcript is stable and ribosome-bound. (A) An McC-producing culture of E. coli harboring the pp70 plasmid was treated with rifampicin. At times indicated, aliquots of the culture were withdrawn, total RNA was purified and amounts of mccA and mccB transcripts were measured by Northern blotting and primer extension, respectively, as described in Figure 2B legend. The results were quantified and the mean values and standard deviations in mRNA abundance obtained in three independent experiments are presented. (B) Lysates of McC-producing cells were subjected to sucrose gradient centrifugation, fractions were collected and the amounts of mccA RNA were determined by Northern blotting. Gradient profile of sucrose centrifugation is shown below the gel. (C) Total RNA was purified from McC-producing E. coli culture harboring the pp70 plasmid or from equally grown cultures harboring pp70 derivatives with a mutated mccA gene initiating codon (ATG1->TAA) or multiple substitutions in the SD sequence of mccA and amounts of the mccA transcript (top), mccB transcript (middle) or bla transcript (bottom) were determined by Northern blot (mccA) or primer extension (mccB, bla) using appropriate primers.
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Figure 3: The mccA transcript is stable and ribosome-bound. (A) An McC-producing culture of E. coli harboring the pp70 plasmid was treated with rifampicin. At times indicated, aliquots of the culture were withdrawn, total RNA was purified and amounts of mccA and mccB transcripts were measured by Northern blotting and primer extension, respectively, as described in Figure 2B legend. The results were quantified and the mean values and standard deviations in mRNA abundance obtained in three independent experiments are presented. (B) Lysates of McC-producing cells were subjected to sucrose gradient centrifugation, fractions were collected and the amounts of mccA RNA were determined by Northern blotting. Gradient profile of sucrose centrifugation is shown below the gel. (C) Total RNA was purified from McC-producing E. coli culture harboring the pp70 plasmid or from equally grown cultures harboring pp70 derivatives with a mutated mccA gene initiating codon (ATG1->TAA) or multiple substitutions in the SD sequence of mccA and amounts of the mccA transcript (top), mccB transcript (middle) or bla transcript (bottom) were determined by Northern blot (mccA) or primer extension (mccB, bla) using appropriate primers.

Mentions: The data presented above can be explained by either transcription termination or transcript processing between the mccA and the mccB ORFs. A processing event would be expected to generate a new 5′ end that should be detectable by primer extension with oligonucleotides annealing to mccB. No such primer extension product was detected (data not shown), suggesting that transcription termination is likely responsible for the appearance of the short mccA transcript. The high ratio of monocistronic to polycistronic mccA transcripts may result from very efficient transcription termination in the intergenic region or it may be caused by differential transcript stability (a combination of these two effects is also possible). To determine transcript stabilities, McC-producing cells were treated with rifampicin, an inhibitor of transcription initiation, and decay of mcc RNA abundance was followed over time by Northern blotting (for mccA transcript) and primer extension (for polycistronic transcript). The results are presented in Figure 3A. As can be seen, the half-life of polycistronic transcript was several minutes, i.e. comparable to half-lives of most E. coli mRNAs (20). In contrast, the short mccA transcript was exceedingly stable (almost no change in transcript abundance after a 20-min incubation with rifampicin). The exceptional stability of the mccA transcript may be responsible for the abnormally low (∼3%) ratio of TEX-resistant transcripts revealed by dRNA-seq (Figure 2A), since transcripts that persist for a long time could lose their 5′ triphosphate moiety (21).


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)

The mccA transcript is stable and ribosome-bound. (A) An McC-producing culture of E. coli harboring the pp70 plasmid was treated with rifampicin. At times indicated, aliquots of the culture were withdrawn, total RNA was purified and amounts of mccA and mccB transcripts were measured by Northern blotting and primer extension, respectively, as described in Figure 2B legend. The results were quantified and the mean values and standard deviations in mRNA abundance obtained in three independent experiments are presented. (B) Lysates of McC-producing cells were subjected to sucrose gradient centrifugation, fractions were collected and the amounts of mccA RNA were determined by Northern blotting. Gradient profile of sucrose centrifugation is shown below the gel. (C) Total RNA was purified from McC-producing E. coli culture harboring the pp70 plasmid or from equally grown cultures harboring pp70 derivatives with a mutated mccA gene initiating codon (ATG1->TAA) or multiple substitutions in the SD sequence of mccA and amounts of the mccA transcript (top), mccB transcript (middle) or bla transcript (bottom) were determined by Northern blot (mccA) or primer extension (mccB, bla) using appropriate primers.
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Figure 3: The mccA transcript is stable and ribosome-bound. (A) An McC-producing culture of E. coli harboring the pp70 plasmid was treated with rifampicin. At times indicated, aliquots of the culture were withdrawn, total RNA was purified and amounts of mccA and mccB transcripts were measured by Northern blotting and primer extension, respectively, as described in Figure 2B legend. The results were quantified and the mean values and standard deviations in mRNA abundance obtained in three independent experiments are presented. (B) Lysates of McC-producing cells were subjected to sucrose gradient centrifugation, fractions were collected and the amounts of mccA RNA were determined by Northern blotting. Gradient profile of sucrose centrifugation is shown below the gel. (C) Total RNA was purified from McC-producing E. coli culture harboring the pp70 plasmid or from equally grown cultures harboring pp70 derivatives with a mutated mccA gene initiating codon (ATG1->TAA) or multiple substitutions in the SD sequence of mccA and amounts of the mccA transcript (top), mccB transcript (middle) or bla transcript (bottom) were determined by Northern blot (mccA) or primer extension (mccB, bla) using appropriate primers.
Mentions: The data presented above can be explained by either transcription termination or transcript processing between the mccA and the mccB ORFs. A processing event would be expected to generate a new 5′ end that should be detectable by primer extension with oligonucleotides annealing to mccB. No such primer extension product was detected (data not shown), suggesting that transcription termination is likely responsible for the appearance of the short mccA transcript. The high ratio of monocistronic to polycistronic mccA transcripts may result from very efficient transcription termination in the intergenic region or it may be caused by differential transcript stability (a combination of these two effects is also possible). To determine transcript stabilities, McC-producing cells were treated with rifampicin, an inhibitor of transcription initiation, and decay of mcc RNA abundance was followed over time by Northern blotting (for mccA transcript) and primer extension (for polycistronic transcript). The results are presented in Figure 3A. As can be seen, the half-life of polycistronic transcript was several minutes, i.e. comparable to half-lives of most E. coli mRNAs (20). In contrast, the short mccA transcript was exceedingly stable (almost no change in transcript abundance after a 20-min incubation with rifampicin). The exceptional stability of the mccA transcript may be responsible for the abnormally low (∼3%) ratio of TEX-resistant transcripts revealed by dRNA-seq (Figure 2A), since transcripts that persist for a long time could lose their 5′ triphosphate moiety (21).

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