Transcription start site sequence and spacing between the -10 region and the start site affect reiterative transcription-mediated regulation of gene expression in Escherichia coli

Abstract

Reiterative transcription is a reaction catalyzed by RNA polymerase, in which nucleotides are repetitively added to the 3' end of a nascent transcript due to upstream slippage of the transcript without movement of the DNA template. In Escherichia coli, the expression of several operons is regulated through mechanisms in which high intracellular levels of UTP promote reiterative transcription that adds extra U residues to the 3' end of a nascent transcript during transcription initiation. Immediately following the addition of one or more extra U residues, the nascent transcripts are released from the transcription initiation complex, thereby reducing the level of gene expression. Therefore, gene expression can be regulated by internal UTP levels, which reflect the availability of external pyrimidine sources. The magnitude of gene regulation by these mechanisms varies considerably, even when control mechanisms are analogous. These variations apparently are due to differences in promoter sequences. One of the operons regulated (in part) by UTP-sensitive reiterative transcription in E. coli is the carAB operon, which encodes the first enzyme in the pyrimidine nucleotide biosynthetic pathway. In this study, we used the carAB operon to examine the effects of nucleotide sequence at and near the transcription start site and spacing between the start site and -10 region of the promoter on reiterative transcription and gene regulation. Our results indicate that these variables are important determinants in establishing the extent of reiterative transcription, levels of productive transcription, and range of gene regulation.

FIG 1

Sequences of the pyrBI, carAB P1, and gal P2 promoter regions from the −10 region through the initially transcribed region. The −10 regions are labeled and underlined, and the transcription start sites are in boldface and marked with an asterisk.

FIG 2

Sequences of carAB promoter P1 mutations used to assess the effects of transcription start site sequence and spacing between the start site and −10 region on reiterative transcription and gene regulation. The wild-type carAB promoter P1 sequence is shown and marked as described for Fig. 1, and arrows indicate the mutations introduced into this sequence. In two mutant promoters designed to examine start site sequence, the wild-type G start site was changed to either A or AA. In two other mutant promoters designed to examine the effects of spacing, either a C was inserted before the wild-type start site or the wild-type G start site was changed to CA.

FIG 3

Comparison of reiterative transcription at the wild-type carAB P1 promoter and mutant promoters with changes in the sequence at the transcription start site. DNA templates containing the wild-type (wt) carAB P1 promoter (lanes 1 and 2) and the G-to-A (lanes 3 and 4) and G-to-AA (lanes 5 and 6) mutant carAB P1 promoters were transcribed in vitro in reaction mixtures containing 400 μM (each) ATP, CTP, GTP, and either 500 or 50 μM UTP, as indicated. Transcripts were radiolabeled at their 5′ ends with either [γ-³²P]GTP or [γ-³²P]ATP, as indicated, separated by polyacrylamide gel electrophoresis, and visualized by autoradiography. The lengths (in nucleotides) of transcripts initiated at the wild-type and mutant promoters are indicated at the left and right sides of the autoradiogram, respectively. Arrows indicate transcripts produced by simple abortive initiation: filled arrows indicate transcripts initiated at the wild-type promoter, and the open arrow indicates the AAUUUG transcript initiated at the G-to-AA promoter. Full-length transcripts are enclosed by a bracket and labeled; heterogeneity in the length of these transcripts is due to termination at multiple sites within the downstream intrinsic terminator (12).

FIG 4

Levels of productive carAp₁::lacZ transcripts initiated in vivo at the wild-type carAB P1 promoter and mutant promoters with changes in the sequence at the transcription start site. Cellular RNA was quantitatively isolated from exponentially growing cells of strains CLT5174 (wild type), CLT5189 (G to A), and CLT5190 (G to AA) grown in the presence of either uracil (R) or UMP (M) (i.e., conditions of pyrimidine excess or limitation, respectively), and transcript levels were measured by primer extension mapping as described in Materials and Methods. The figure shows an autoradiogram of a gel used to separate and analyze primer extension products. A dideoxy sequencing ladder (i.e., the four lanes marked G, A, T, and C) of the wild-type promoter region, which was used to identify both wild-type and mutant transcripts, was produced with the same DNA primer as that used for primer extension mapping. (Note that the sequence shown on the left is that of the template DNA strand, which is complementary to the sequence of the wild-type carAp₁::lacZ transcript.) Primer extension product levels, which correspond to transcript levels, were measured with a PhosphorImager. Relative levels of transcripts and fold pyrimidine-mediated regulation are indicated.

FIG 5

Comparison of reiterative transcription at wild-type and mutant carAB P1 promoters with different spacing between the −10 region and the transcription start site. DNA templates containing the wild-type (wt) carAB P1 promoter (lanes 1 and 2) and the +C (lanes 3 and 4), G-to-CA (lanes 5 and 6), and G-to-A (lanes 7 and 8) mutant carAB P1 promoters were transcribed in vitro, ³²P labeled at their 5′ ends, and analyzed as described in the legend to Fig. 3. All lanes were from the same autoradiogram, but lanes 7 and 8 were repositioned to facilitate comparison to lanes 5 and 6. The lengths (in nucleotides) of GUUU_n transcripts initiated at the wild-type and +C mutant promoters are indicated on the left, with arrows indicating transcripts produced by simple abortive initiation at the two promoters. The lengths of AUUU_n transcripts initiated on the G-to-CA and G-to-A mutant promoters are indicated at the right. The [γ-³²P]NTP used to label the 5′ ends of transcripts is indicated at the bottom, and full-length transcripts are enclosed by a bracket and labeled.

FIG 6

Levels of productive carAp₁::lacZ transcripts initiated in vivo at wild-type and mutant carAB P1 promoters with different spacing between the −10 region and transcription start site. Cellular RNA was quantitatively isolated from exponentially growing cells of strains CLT5174 (wild type), CLT5237 (+C), and CLT5238 (G to CA) grown in the presence of either uracil (R) or UMP (M), and transcript levels were analyzed by primer extension mapping as described above. The primer extension mapping data for strain CLT5189 (G to A) were taken from Fig. 4; the analysis of the data in Fig. 4 and 6 was identical. The dideoxy sequencing ladder (lanes G, A, T, and C) of the wild-type promoter region was generated as described in the legend to Fig. 4. Primer extension product levels, which correspond to transcript levels, were measured with a PhosphorImager, and the relative levels of transcripts and fold pyrimidine-mediated regulation are indicated.