Ribosome-binding sites and RNA-processing sites in the transcript of the Escherichia coli unc operon

The polycistronic mRNA encoding the nine genes of the unc operon of Escherichia coli was studied. We demonstrated the ribosome-binding capabilities of six of the nine unc genes, uncB, uncE, uncF, uncH, uncA, and uncD, by using the technique of primer extension inhibition or "toeprinting." No toeprint was detected for the other genes, uncI, uncG, and uncC. The lack of a toeprint for uncG suggests that this gene is expressed by some form of translational coupling, such that either uncG is read by ribosomes which have translated the preceding gene, uncA, or translation of uncA is required for ribosome binding at the uncG site. RNA sequencing and primer extension in the regions of uncI and uncC, the first and last genes in the operon, respectively, gave less intense signals than those obtained for the other unc genes. This suggested that there are fewer copies of those regions of the transcript and that processing of the unc transcript occurred. Using primer extension and RNA sequencing, we identified sites in the unc transcript at which processing appears to take place, including a site which may remove much of the uncI portion of the transcript. Northern (RNA) blot analysis of unc RNA is consistent with the presence of an RNA-processing site in the uncI region of the transcript and another in the uncH region. These processing events may account for some of the differential levels of expression of the unc genes.

[1]  D. Klionsky,et al.  Escherichia coli mutants defective in the uncH gene , 1983, Journal of bacteriology.

[2]  D. Klionsky,et al.  In vivo evidence for the role of the epsilon subunit as an inhibitor of the proton-translocating ATPase of Escherichia coli , 1984, Journal of bacteriology.

[3]  M. Futai,et al.  Structure and function of proton-translocating adenosine triphosphatase (F0F1): biochemical and molecular biological approaches. , 1983, Microbiological reviews.

[4]  L. Gold,et al.  Extension inhibition analysis of translation initiation complexes. , 1988, Methods in enzymology.

[5]  J. Shine,et al.  The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. , 1974, Proceedings of the National Academy of Sciences of the United States of America.

[6]  K. Solomon,et al.  Effect of an uncE ribosome-binding site mutation on the synthesis and assembly of the Escherichia coli proton-translocating ATPase. , 1988, The Journal of biological chemistry.

[7]  R. Gunsalus,et al.  Gene order and gene-polypeptide relationships of the proton-translocating ATPase operon (unc) of Escherichia coli. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[8]  W. Sebald,et al.  Translational initiation frequency of atp genes from Escherichia coli: identification of an intercistronic sequence that enhances translation. , 1985, The EMBO journal.

[9]  D. Klionsky,et al.  Differential translation of the genes encoding the proton-translocating ATPase of Escherichia coli. , 1986, The Journal of biological chemistry.

[10]  J. McCarthy,et al.  Determinants of translational initiation efficiency in the atp operon of Escherichia coli , 1988, Molecular microbiology.

[11]  L. Gold,et al.  Bacteriophage T4 regA protein binds to mRNAs and prevents translation initiation. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[12]  D. Klionsky,et al.  Differential polypeptide synthesis of the proton-translocating ATPase of Escherichia coli , 1982, Journal of bacteriology.

[13]  T. Noumi,et al.  Nucleotide sequence of the genes for F0 components of the proton-translocating ATPase from Escherichia coli: prediction of the primary structure of F0 subunits. , 1981, Biochemical and biophysical research communications.

[14]  R. Traut,et al.  The subunit interface of the Escherichia coli ribosome. Crosslinking of 30 S protein S9 to proteins of the 50 S subunit. , 1979, Journal of molecular biology.

[15]  H. Noller,et al.  Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. , 1981, Journal of molecular biology.

[16]  G. Stormo,et al.  CUUCGG hairpins: extraordinarily stable RNA secondary structures associated with various biochemical processes. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[17]  G. Stormo,et al.  Translational regulation of expression of the bacteriophage T4 lysozyme gene. , 1986, Nucleic acids research.

[18]  J. Walker,et al.  The unc operon. Nucleotide sequence, regulation and structure of ATP-synthase. , 1984, Biochimica et biophysica acta.

[19]  M. Futai,et al.  Nucleotide sequence of the genes for β and ε subunits of proton-translocating ATPase from Escherichia coli , 1982 .

[20]  G. Stormo,et al.  Autogenous regulatory site on the bacteriophage T4 gene 32 messenger RNA. , 1988, Journal of molecular biology.

[21]  J. Steitz,et al.  How ribosomes select initiator regions in mRNA: base pair formation between the 3' terminus of 16S rRNA and the mRNA during initiation of protein synthesis in Escherichia coli. , 1975, Proceedings of the National Academy of Sciences of the United States of America.

[22]  J. McCarthy,et al.  Post-transcriptional control in Escherichia coli: translation and degradation of the atp operon mRNA. , 1988, Gene.

[23]  R. Young,et al.  Dominance in lambda S mutations and evidence for translational control. , 1988, Journal of molecular biology.