Role of the ribosome in suppressing transcriptional termination at the pyrBI attenuator of Escherichia coli K-12

K. L. Roland, C. Liu, C. L. Turnbough

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

Pyrimidine-mediated regulation of pyrBI operon expression in Escherichia coli K-12 occurs primarily by an attenuation control mechanism. Previous studies have suggested a model for attenuation control in which low intracellular levels of UTP cause close coupling of transcription and translation within the pyrBI leader region. This close coupling apparently prevents transcriptional termination at an attenuator (a ρ-independent transcriptional terminator) located 23 base pairs before the pyrBI structural genes within an open reading frame for a 44-amino acid leader polypeptide. Presumably, a ribosome involved in the synthesis of the leader polypeptide disrupts or precludes the formation of the attenuator-encoded RNA hairpin, which is required for transcriptional termination. In this study, we examined the role of the ribosome in inhibiting transcriptional termination at the pyrBI attenuator. Using oligonucleotide-directed mutagenesis, we systematically introduced termination codons into the reading frame for the leader polypeptide to determine the distance a ribosome must translate to suppress transcriptional termination. These mutations were incorporated individually into a pyrB::lacZ gene fusion, which was then introduced into the E. coli chromosome. The resulting fusion strains were used to measure the effect of each mutation on pyrB::lacZ expression. The results show that a ribosome must translate to within 14-16 nucleotides of the attenuator-encoded RNA hairpin to inhibit transcriptional termination efficiently, which indicates a direct interaction between the ribosome and the termination hairpin sequence as proposed in the present model. Additional results indicate that factors not included in the present model for attenuation control contribute to the expression and regulation of the pyrBI operon.

Original languageEnglish (US)
Pages (from-to)7149-7153
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume85
Issue number19
StatePublished - 1988
Externally publishedYes

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Ribosomes
Escherichia coli
Operon
Peptides
RNA
Reading Frames
Mutation
Uridine Triphosphate
Lac Operon
Terminator Codon
Gene Fusion
Site-Directed Mutagenesis
Base Pairing
Open Reading Frames
Nucleotides
Chromosomes
Amino Acids
Genes

ASJC Scopus subject areas

  • General
  • Genetics

Cite this

Role of the ribosome in suppressing transcriptional termination at the pyrBI attenuator of Escherichia coli K-12. / Roland, K. L.; Liu, C.; Turnbough, C. L.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 85, No. 19, 1988, p. 7149-7153.

Research output: Contribution to journalArticle

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AB - Pyrimidine-mediated regulation of pyrBI operon expression in Escherichia coli K-12 occurs primarily by an attenuation control mechanism. Previous studies have suggested a model for attenuation control in which low intracellular levels of UTP cause close coupling of transcription and translation within the pyrBI leader region. This close coupling apparently prevents transcriptional termination at an attenuator (a ρ-independent transcriptional terminator) located 23 base pairs before the pyrBI structural genes within an open reading frame for a 44-amino acid leader polypeptide. Presumably, a ribosome involved in the synthesis of the leader polypeptide disrupts or precludes the formation of the attenuator-encoded RNA hairpin, which is required for transcriptional termination. In this study, we examined the role of the ribosome in inhibiting transcriptional termination at the pyrBI attenuator. Using oligonucleotide-directed mutagenesis, we systematically introduced termination codons into the reading frame for the leader polypeptide to determine the distance a ribosome must translate to suppress transcriptional termination. These mutations were incorporated individually into a pyrB::lacZ gene fusion, which was then introduced into the E. coli chromosome. The resulting fusion strains were used to measure the effect of each mutation on pyrB::lacZ expression. The results show that a ribosome must translate to within 14-16 nucleotides of the attenuator-encoded RNA hairpin to inhibit transcriptional termination efficiently, which indicates a direct interaction between the ribosome and the termination hairpin sequence as proposed in the present model. Additional results indicate that factors not included in the present model for attenuation control contribute to the expression and regulation of the pyrBI operon.

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