Dataset Information

A divergently transcribed open reading frame is located upstream of the Pseudomonas aeruginosa vfr gene, a homolog of Escherichia coli crp.

ABSTRACT: The Pseudomonas aeruginosa homolog of the Escherichia coli global transcriptional regulator CRP (or CAP) was recently identified and designated Vfr (S. E. H. West, A. K. Sample, and L. J. Runyen-Janecky, J. Bacteriol. 176:7532-7542, 1994). Nucleotide sequence analysis of the region 5' to vfr identified a 423-bp open reading frame (ORF), which was designated orfX. The deduced amino acid sequence of ORFX was 53% identical and 87% similar to a divergent ORF of unknown function located 5' to the E. coli crp gene. When orfX was expressed from a phage T7 promoter in E. coli, a protein with an apparent molecular mass of approximately 18 kDa was produced. We constructed a chromosomal deletion of the region containing the 5' end of orfX (orfX'), vfr, and the 3' end of trpC (trpC') in P. aeruginosa strains PAO1 and PA103. The cloned vfr gene restored Vfr-dependent production of exotoxin A and protease in the PA103 orfX'-vfr-trpC' deletion mutant, suggesting that ORFX is not required for Vfr production or activity. To determine whether transcription of orfX and vfr are controlled by the same mechanisms that control transcription of the region of the divergent ORF (dorf) and of crp, we compared the vfr-orfX and crp-dorf intergenic regions. Using S1 nuclease analysis, we determined that the distance between the orfX and vfr transcriptional start sites was 105 bp. Thus, the P. aeruginosa orfX and vfr promoters are arranged in a back-to-back orientation rather than the face-to-face orientation of the dorf and crp promoters. A CRP recognition site is associated with each promoter in the crp-dorf intergenic region; binding of the CRP-cyclic AMP complex to the stronger dorf CRP recognition site activates transcription from the dorf promoter and represses transcription from the crp promoter. The vfr-orfX intergenic region does not contain an obvious CRP recognition site. In addition, vfr was not required for transcription of orfX. Unlike the dorf and crp mRNAs, the 5' ends of the orfX and vfr mRNAs were not complementary. Thus, the orfX mRNA cannot hybridize to the 5' end of the vfr mRNA to inhibit vfr transcription, a mechanism that has been postulated to control crp transcription in E. coli.

SUBMITTER: Runyen-Janecky LJ

PROVIDER: S-EPMC179038 | biostudies-other | 1997 May

REPOSITORIES: biostudies-other

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A divergently transcribed open reading frame is located upstream of the Pseudomonas aeruginosa vfr gene, a homolog of Escherichia coli crp.

Runyen-Janecky L J LJ Sample A K AK Maleniak T C TC West S E SE

Journal of bacteriology 19970501 9

The Pseudomonas aeruginosa homolog of the Escherichia coli global transcriptional regulator CRP (or CAP) was recently identified and designated Vfr (S. E. H. West, A. K. Sample, and L. J. Runyen-Janecky, J. Bacteriol. 176:7532-7542, 1994). Nucleotide sequence analysis of the region 5' to vfr identified a 423-bp open reading frame (ORF), which was designated orfX. The deduced amino acid sequence of ORFX was 53% identical and 87% similar to a divergent ORF of unknown function located 5' to the E. ...[more]

PMID: 9139892

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Project description:Translation of an mRNA in eukaryotes starts at AUG in most cases. Near-cognate codons (NCCs) such as UUG, ACG and AUU are also used as start sites at low levels in S. cerevisiae. Initiation from NCCs or AUGs in the 5’-untranslated regions (UTRs) of mRNAs can lead to translation of upstream open reading frames (uORFs) that might regulate expression of the main ORF (mORF). Although there is some circumstantial evidence that the translation of uORFs can be affected by environmental conditions, little is known about how it is affected by changes in growth temperature. Using reporter assays, we found that changes in growth temperature can affect translation from NCC start sites in yeast cells, suggesting the possibility that gene expression could be regulated by temperature by altering use of different uORF start codons. Using ribosome profiling, we provide evidence that growth temperature regulates the efficiency of translation of nearly 200 uORFs in S. cerevisiae. Of these uORFs, most that start with an AUG codon have increased translational efficiency at 37 ˚C relative to 30 ˚C and decreased efficiency at 20 ˚C. For translationally regulated uORFs starting with NCCs, we did not observe a general trend for the direction of regulation as a function of temperature, suggesting mRNA-specific features can determine the mode of temperature-dependent regulation. Consistent with this conclusion, the position of the uORFs in the 5’-leader relative to the 5’-cap and the start codon of the main ORF correlates with the direction of temperature-dependent regulation of uORF translation. We have identified several novel cases in which changes in uORF translation are inversely correlated with changes in the translational efficiency of the downstream main ORF. Our data suggest that translation of these mRNAs is subject to temperature-dependent, uORF-mediated regulation. Overall, our data suggest that alterations in the translation of specific uORFs by temperature can regulate gene expression in S. cerevisiae.

Project description:BACKGROUND:Translation of an mRNA in eukaryotes starts at an AUG codon in most cases, but near-cognate codons (NCCs) such as UUG, ACG, and AUU can also be used as start sites at low levels in Saccharomyces cerevisiae. Initiation from NCCs or AUGs in the 5'-untranslated regions (UTRs) of mRNAs can lead to translation of upstream open reading frames (uORFs) that might regulate expression of the main ORF (mORF). Although there is some circumstantial evidence that the translation of uORFs can be affected by environmental conditions, little is known about how it is affected by changes in growth temperature. RESULTS:Using reporter assays, we found that changes in growth temperature can affect translation from NCC start sites in yeast cells, suggesting the possibility that gene expression could be regulated by temperature by altering use of different uORF start codons. Using ribosome profiling, we provide evidence that growth temperature regulates the efficiency of translation of nearly 200 uORFs in S. cerevisiae. Of these uORFs, most that start with an AUG codon have increased translational efficiency at 37 °C relative to 30 °C and decreased efficiency at 20 °C. For translationally regulated uORFs starting with NCCs, we did not observe a general trend for the direction of regulation as a function of temperature, suggesting mRNA-specific features can determine the mode of temperature-dependent regulation. Consistent with this conclusion, the position of the uORFs in the 5'-leader relative to the 5'-cap and the start codon of the main ORF correlates with the direction of temperature-dependent regulation of uORF translation. We have identified several novel cases in which changes in uORF translation are inversely correlated with changes in the translational efficiency of the downstream main ORF. Our data suggest that translation of these mRNAs is subject to temperature-dependent, uORF-mediated regulation. CONCLUSIONS:Our data suggest that alterations in the translation of specific uORFs by temperature can regulate gene expression in S. cerevisiae.

Dataset Information

A divergently transcribed open reading frame is located upstream of the Pseudomonas aeruginosa vfr gene, a homolog of Escherichia coli crp.

Publications

A divergently transcribed open reading frame is located upstream of the Pseudomonas aeruginosa vfr gene, a homolog of Escherichia coli crp.

Similar Datasets

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