Translation of galE and coordination of galactose operon expression in Escherichia coli: effects of insertions and deletions in the non‐translated leader sequence

Abstract

Using gene-manipulation techniques, we made a set of short insertions and deletions in the Escherichia coli galactose operon between the transcription start site and the Shine-Dalgarno sequence of the first gene of the operon, galE. Translation initiation is severely reduced when the distance between the 5' end of the message and the Shine-Dalgarno sequence drops below 15 bases. Transcription of the gal operon can start at two distinct sites, S1 and S2, separated by 5 bp, situated 16 and 21 bp upstream of the galE Shine-Dalgarno sequence, respectively. When transcription starts at S2, gal operon expression is discoordinate as the galE gene is better translated than promoter-distal genes. Here we report that gal operon expression is discoordinate even when message starting at S2 is shortened. This shows that the better translation of galE from transcripts starting at S2 is not simply due to the fact that they are longer than transcripts starting at S1.

Organization of the LEE1 operon regulatory region of enterohaemorrhagic Escherichia coli O157:H7 and activation by GrlA

“…Plasmid pRW224 is an RK2‐based low‐copy‐number lac expression vector, encoding resistance to tetracycline, designed to facilitate the cloning of EcoRI–HindIII fragments carrying a promoter directed towards the HindIII end of the fragment but without a translation start. Thus, a translation initiation signal for lacZ is located in the vector immediately adjacent to the HindIII site as described by Bingham and Busby (1987) and in Fig. S1.…”

“…C. GrlA interacts with the spacer sequence and facilitates RNA polymerase activity. located in the vector immediately adjacent to the HindIII site as described by Bingham and Busby (1987) and in Fig. S1.…”

Attempts to engineer an Escherichia coli DNA-binding protein as a tool for affinity purification of heterologous proteins

“…Synthetic oligodeoxyribonucleotides were made by Alta Biochemicals (University of Birmingham). The BumHI-SuZI fragment carrying the entire Bgalactosidase gene was obtained from pAA (Bingham and Busby, 1987). The HindIIIEcoFU fragment encoding the human Ck domain was obtained from plasmid pRB32 (Whittle et al, 1987).…”

Hypersymmetry in a transcriptional terminator of Escherichia coli confers increased efficiency as well as bidirectionality.

“…We therefore constructed a new vector, pJW30 ( Figure 3A), in which galK is served by a Pgal promoter fragment from which the entire galE ribosome binding site has been deleted (and the CAP-dependent P1 promoter has incidentally been inactivated, by a G to A mutation at 'minus 14'). pJW30 was constructed by replacing the EcoRI-HindIIl (Pgal) fragment of pHR9 with the corresponding fragment of pAA204 p14 1 12 (Bingham and Busby, 1987). We are much indebted to Dr S.Busby both for providing this plasmid and for suggesting its use.…”