Transcription regulatory elements are punctuation marks for DNA replication

Abstract

Collisions between DNA replication and transcription significantly affect genome organization, regulation, and stability. Previous studies have described collisions between replication forks and elongating RNA polymerases. Although replication collisions with the transcription-initiation or -termination complexes are potentially even more important because most genes are not actively transcribed during DNA replication, their existence and mechanisms remained unproven. To address this matter, we have designed a bacterial promoter that binds RNA polymerase and maintains it in the initiating mode by precluding the transition into the elongation mode. By using electrophoretic analysis of replication intermediates, we have found that this steadfast transcription-initiation complex inhibits replication fork progression in an orientation-dependent manner during head-on collisions. Transcription terminators also appeared to attenuate DNA replication, but in the opposite, codirectional orientation. Thus, transcription regulatory signals may serve as "punctuation marks" for DNA replication in vivo.

Fig. 1.

AT-rich ITS abolishes promoter activity. (A) Nucleotide sequences of the original bacteriophage T7 early promoter A1 and its mutant derivative, in which the region from +1 to +20 became 90% AT-rich promoter B. The −35, −10, and +1 positions are underlined; the AT-rich stretch is highlighted. (B) In vitro transcription assay on the templates of the promoters A and B. The position of the anticipated 25-nt-long product is marked by an arrow. (C) Northern blot analysis of the transcriptional activity of the promoters A and B in vivo. Only informative portions of the corresponding autoradiographs are shown in Left. (Right) Quantification of the results. HO, head-on orientation of the promoter to the direction of replication in the plasmids; CD, codirectional orientation. Maps of the plasmids pA-HO, pB-HO, pA-CD, and pB-CD are shown in Figs. 3A and 4A. TR, promoter-specific transcript; RNA I, plasmid-derived RNA used for the normalization. (D) Chemical probing of the open-complex formation in situ. Two different primers were used to detect modifications of the template (right half) and nontemplate (left half) strands in promoters A and B (pA-HO and pB-HO plasmids, respectively). Asterisks, primer extension reactions on the in situ chloroacetaldehyde-modified templates; G, A, T, and C, Sanger sequencing reactions on control templates with the same primers. The −10 and +1 positions of the promoters are marked.

Fig. 2.

Replication stalling by the head-on oriented transcription-initiation complex. (A Left) Map of the plasmids pA-HO and pB-HO. Transcription cassettes with promoters A and B, respectively, are oriented head-on to the direction of replication; nucleotide position 1 in the plasmids corresponds to the replication start site; positions of the promoters and T1T2 terminators are shown. (A Right) Schematic representation of the two-dimensional gel electrophoresis of replication intermediates. The bubble arc consists of replication intermediates, starting from the small bubble at the origin of replication (at the bottom) and going up to the biggest, fully replicated bubble (at the top). Replication stalling is detected as a thickening of a particular segment of the arc. P, promoter; T, terminator; arrow, direction of transcription. (B) Two-dimensional gel electrophoresis of replication intermediates. Replication stalling is evident as a long segment that corresponds to the whole transcribed area in the pA-HO plasmid (Left) or as a bulge that corresponds to the promoter in the pB-HO plasmid (Right). (C) Quantitative analysis of the replication arcs around the transcribed units in plasmids pA-HO and pB-HO. The experiment shown here is independent of the one shown in Fig. 4B. (D) Scheme for mapping replication pause sites by in-gel digestion of replication intermediates (for details, see Results). (Left) The vertical lines show the positions of the restriction sites immediately upstream (PstI) and downstream (EcoRI) from the promoter (P). Replication intermediates are shown as bubbles, and the bold bubble corresponds to stalled replication intermediates. (Center and Right) During EcoRI digestion (Center), stalled intermediates become Y-shaped and move to the line, whereas after PstI digestion (Right), they remain bubble-shaped and stay on the arc. (E) Experimental mapping of stalled replication intermediates by in-gel digestion with EcoRI (Left) or PstI (Right). Stalled replication intermediates are shown by arrows. Note the underreplicated stalled intermediates migrating between the arc and the line (for details, see Results).

Fig. 3.

Replication stalling by the codirectionally oriented transcription terminators. (A Left) Map of plasmids pA-CD and pB-CD. Transcription cassettes with promoters A or B, respectively, are oriented codirectionally with the direction of replication; nucleotide position 1 corresponds to the replication start site; positions of the promoters and T1T2 terminators are indicated. (A Right) Schematic representation of the two-dimensional gel electrophoresis of replication intermediates. Unlike Fig. 2, a transcription cassette faces up, i.e., away from the origin. (B) Two-dimensional gel electrophoresis of replication intermediates. Replication stalling is evident in the pA-CD plasmid at the position that corresponds to the position of the transcription terminators (arrow). (C). A scheme of mapping stalled intermediates by in-gel digestion is shown. Replication intermediates were separated in the first dimension followed by digestion with the XbaI and PstI enzymes, flanking the T1T2 terminators (T). (D) Experimental mapping of stalled intermediates. During XbaI digestion (Left), stalled intermediates become Y-shaped and move to the line, whereas during PstI digestion (Right), they remain bubble-shaped and stay on the arc.

Fig. 4.

A model of transcription regulatory elements, serving as punctuation marks for DNA replication. P, promoter; T, terminator. For details, see Discussion.