The distribution of active RNA polymerase II along the transcribed region is gene-specific and controlled by elongation factors

Abstract

In order to study the intragenic profiles of active transcription, we determined the relative levels of active RNA polymerase II present at the 3'- and 5'-ends of 261 yeast genes by run-on. The results obtained indicate that the 3'/5' run-on ratio varies among the genes studied by over 12 log(2) units. This ratio seems to be an intrinsic characteristic of each transcriptional unit and does not significantly correlate with gene length, G + C content or level of expression. The correlation between the 3'/5' RNA polymerase II ratios measured by run-on and those obtained by chromatin immunoprecipitation is poor, although the genes encoding ribosomal proteins present exceptionally low ratios in both cases. We detected a subset of elongation-related factors that are important for maintaining the wild-type profiles of active transcription, including DSIF, Mediator, factors related to the methylation of histone H3-lysine 4, the Bur CDK and the RNA polymerase II subunit Rpb9. We conducted a more detailed investigation of the alterations caused by rpb9Delta to find that Rpb9 contributes to the intragenic profiles of active transcription by influencing the probability of arrest of RNA polymerase II.

Figure 1.

(A) Average 3′/5′ ratios of the run-on signals obtained from a wild-type BY4741 strain for the transcriptional units present in the 377 genes array. RP genes are represented in red and RiBi genes in green. Error bars represent standard deviation. The box-and-whiskers diagrams represent the minimum, the maximum, the average (cross), the median and the 25th and the 75th percentiles of the average 3′/5′ run-on ratios for the indicated gene categories. The P-values of the Student’s two-tailed t-test for those samples with unequal variances are shown. (B) The same as in A but for the ChIP signals obtained with the 8WG16 antibody. (C) Scatter plot of the average 3′/5′ run-on ratios in A (X-axis) versus the average 3′/5′ ChIP ratios in B (Y-axis). Linear regressions and their corresponding Pearson’s correlation coefficients (R) are shown.

Figure 2.

Box-and-whiskers diagrams representing the increments in the 3′/5′ run-on ratios for the transcription units present in the array of highly expressed genes in the indicated strains and conditions. The diagrams summarize the data represented as scatter plots in the Supplementary Figure S5. Dark gray indicates a significant P-value.

Figure 3.

(A) Image of a typical hybridization of the array of highly expressed genes with a run-on preparation from the BY4741 wild-type strain (top) and an isogenic spt4Δ mutant (bottom). See Supplementary Figure S1 and Supplementary Table SI for probe keys. The signals of six transcriptional units, in which changes in the 3′/5′ ratio between the wild type and the mutant can be easily seen, are squared. (B) Scatter plot of the run-on 3′/5′ ratios obtained for a wild-type BY4741 strain (X-axis) and an spt4Δ mutant (Y-axis). The dashed line represents a perfect match for the X and Y values. The continuous line represents linear regression. The distance between the two lines reflects the average effect of the mutant. P-value for the Student’s two-tailed t-test for paired samples (P) and Pearson’s coefficient (R) are shown. The lower the Pearson’s coefficient of a mutant, the more gene-specific its effect on the 3′/5′ ratios. (C) The same as in (B) but for the ssd1Δ mutant. (D) Box-and-whiskers diagrams representing the increments in the 3′/5′ run-on ratios for the transcription units present in the array of highly expressed genes, in the indicated mutant, related to the corresponding isogenic wild types. The boxes representing the mutants with a significantly higher P-value are shown in dark gray, those with a significantly lower value are represented in light gray, and those with no statistically significant difference are represented in white.

Figure 4.

(A) Scatter plot of the run-on 3′/5′ ratios obtained for a wild-type BY4741 strain (X axis) and an rpb9Δ mutant (Y axis) using the array of 377 genes. The box-and-whiskers diagrams summarize the rpb9Δ/wild-type differences for the genes belonging to the indicated gene categories. The effect of rpb9Δ on RP and RiBi genes, relative to non ribosome-related genes, was challenged with the Student’s two-tails t-test for samples with unequal variances; P-values are shown underneath. The other details are as in Figure 3B. (B) Comparison of the run-on profile (red) and the RNA pol II ChIP profile (blue) of the rpb9Δ mutant (relative to the wild-type profile) in HXK2 and HXT1. Error bars represent standard deviation. (C) The same as in (A) but for the RNA pol II 3′/5′ ratios. (D) Scatter plot of the average run-on and ChIP 3′/5′ ratios of rpb9Δ corresponding to the genes represented in the 377 genes array. Linear regressions and their corresponding Pearson’s coefficients are shown.

Figure 5.

Scatter plot representing the average increment of the 3′/5′ run-on ratio produced on each gene by the 18 mutation tested (Y-axis) against the run-on 3′/5′ ratio exhibited by that gene in the wild-type BY4741 strain (X-axis). Spearmann’s rank correlation ρ = –0,428; P = 8.09 × 10^–4.