Synergistic action of RNA polymerases in overcoming the nucleosomal barrier

Abstract

During gene expression, RNA polymerase (RNAP) encounters a major barrier at a nucleosome and yet must access the nucleosomal DNA. Previous in vivo evidence has suggested that multiple RNAPs might increase transcription efficiency through nucleosomes. Here we have quantitatively investigated this hypothesis using Escherichia coli RNAP as a model system by directly monitoring its location on the DNA via a single-molecule DNA-unzipping technique. When an RNAP encountered a nucleosome, it paused with a distinctive 10-base pair periodicity and backtracked by approximately 10-15 base pairs. When two RNAPs elongate in close proximity, the trailing RNAP apparently assists in the leading RNAP's elongation, reducing its backtracking and enhancing its transcription through a nucleosome by a factor of 5. Taken together, our data indicate that histone-DNA interactions dictate RNAP pausing behavior, and alleviation of nucleosome-induced backtracking by multiple polymerases may prove to be a mechanism for overcoming the nucleosomal barrier in vivo.

Figure 1

Locating an RNAP during elongation on nucleosomal DNA. (a) A cartoon of the transcription elongation complex. Unzipping direction is indicated by a red arrow. (b) An example trace of unzipping DNA through a PTC. RNAP was stalled at the +20 nt position relative to transcription start site. The RNAP unzipping force signature (black) shows a distinctive force drop immediately followed by a sharp force rise. The unzipping force of the corresponding naked DNA is shown for comparison (grey). (c) Location distribution of the unzipping force rise obtained by pooling a number of measurements such as that shown in (b). The dashed line indicates the expected location of the 3′ end of the transcribed RNA. (d) The single-promoter transcription template construct containing both a single T7A1 promoter and a 601 nucleosome positioning element (NPE). (e) An example unzipping trace of a template containing both a PTC stalled at +20 nt and a positioned nucleosome. Unzipping confirmed that the RNAP and the nucleosome were at their expected locations. Two regions of strong histone-DNA interactions in a nucleosome are indicated: Region 1 (off-dyad interactions) and Region 2 (dyad interactions). The brown bar indicates the 147-bp 601 NPE. (f) Representative traces of unzipping through an elongation complex. After transcription was resumed for an indicated duration, it was quenched and histones were dissociated. Unzipping revealed the location of the remaining RNAP. Each trace is from a different DNA molecule. The unzipping force of the corresponding naked DNA is shown for comparison (grey).

Figure 2

Transcription through a nucleosome shows a distinctive 10 bp periodicity pausing pattern. (a) RNAP transcribed through a nucleosome in the forward direction of the 601 NPE as indicated by the template cartoon (identical to Fig. 1d). PAGE analysis of transcription through naked DNA and nucleosomal DNA shows that as RNAP proceeded into the nucleosome, a distinctive periodicity of ~ 10 bp highlighted all nucleosome-induced pause sites within Regions 1 and 2. Transcription pause sites are marked as distances from the dyad. (b) RNAP transcribed through a nucleosome from the reverse direction of 601 NPE as indicated by the template cartoon. Although RNAP effectively transcribed a different sequence, all nucleosome-induced pauses were again highlighted by a distinctive ~ 10 bp periodicity within Regions 1 and 2. The pause site at the end of the 601 NPE might be intrinsic pausing (compare transcription through naked DNA and nucleosomal DNA). Also note that at this pause site the leading edge of the RNAP was ~ 20 bp downstream of the 601 NPE.

Figure 3

Histone-DNA interactions induce RNAP backtracking and prevention of backtracking facilitates transcription. All experiments were conducted using the single-promoter DNA template and for 10 s transcription time. The predominant peak position in each distribution is indicated by an arrow. (a) A cartoon of a backtracked transcription elongation complex. Pink dashed line indicates the location of the 3′ end of RNA, and the purple dashed line indicates the location of RNAP active site. (b) An intensity scan of the gel shown in Figure 2a. The 3’ RNA location is specified relative to the dyad. (c) Distribution of RNAP active site location as determined by the unzipping method. The active site location is specified relative to the dyad. The displacement between the peak location of the active site and that of the 3′ end of the RNA indicates the backtracking distance. (d) Distribution of RNAP active site location in the presence of RNase T1.

Figure 4

Trailing RNAP assists leading RNAP to exit an arrested state. (a) The two-promoter transcription template construct contains two T7A1 promoters followed by a single 601 NPE. (b) Example unzipping trace from the template shown in (a) containing two PTCs at their respective +20 nt positions. The two RNAPs were detected at their expected locations. (c) Percentage of RNAP that remained near the +20 nt position versus transcription time for leading and trailing RNAPs. The inset more clearly shows the percentage of the RNAP remaining near the +20 nt.

Figure 5

Two RNAPs work synergistically to overcome a nucleosomal barrier. (a) The two-promoter transcription template construct contains two T7A1 promoters followed by a single 601 NPE (same as Fig. 4a). (b) Example unzipping trace from the template shown in (a) containing two PTCs at their respective +20 bp positions and a positioned nucleosome before transcription resumption. The two RNAPs and the nucleosome were detected at their expected locations. The brown bar indicates the 147-bp 601 NPE. (c) Representative unzipping traces through two elongation complexes on a single DNA molecule after transcription for the indicated durations and after removal of histones. Each trace was from a different DNA molecule. Both the leading and trailing RNAPs were detected by their unzipping signatures. (d) Distribution of the leading RNAP active site location after 10 s transcription reaction.

Figure 6

Transcription efficiency comparison and cartoon illustrating the mechanism of transcription through nucleosomal DNA. (a) Transcription runoff efficiencies vs. transcription time. A runoff efficiency was represented by the percentage of DNA template that showed an absence of RNAP during DNA unzipping experiments. The error bars are standard errors of the means. Smooth curves passing through the data points for each transcription condition were drawn for ease of comparison (not fits). Naked DNA runoff efficiency (black) was obtained from PAGE gel analysis and is shown for comparison. (b) Bar plot of relative transcription rates through nucleosomal DNA. The initial rate of a single RNAP transcribing through a nucleosomal template is used as a reference. The initial rates were estimated from the slopes of linear fits to the near zero transcription times (≤ 1 min). Note that since the trailing promoter is about 162 bp upstream of the leading promoter, a 10 s time delay was taken into account for the trailing RNAP transcription rate calculation. The 50% runoff rate is the reciprocal of the time to achieve 50% runoff. (c–g) Cartoon illustrations of the mechanism of transcription through a nucleosome. As an RNAP approaches a nucleosome (c), it encounters histone-DNA interactions in a nucleosome which induce RNAP pausing and likely backtracking (d and e). The arrival of a trailing RNAP (f) exerts an assisting force on the leading RNAP, rescuing the leading RNAP from its backtracked state. The two RNAPs, working synergistically, eventually evict downstream histones, resulting in the removal of the nucleosomal barrier and the resumption of efficient transcription (g). Regions of strong histone-DNA interactions in the nucleosomal DNA are indicated in red and pink.