Indeed, over the entire range of codons from position 16 to 60, we find that 69% of membrane proteins have at least one programmed pause position, significantly higher compared to cytosolic (50%, p = 1.5 × 10−7) and periplasmic proteins (41%, p = 6 × 10−8, Z-test for 2 proportions).
To assign precise pause positions, we also compared pause bands to 3′-deoxyNTP-generated ladders using shorter templates and high-resolution gels.
Play/Pause: allows you to pause a song and play a song from the beginning or any paused position.
For example, reduction of the pause at position 17 may be caused by introduction of T A base pair five positions upstream of the pause site, which, according to our results, would destabilize EC17WT.
In contrast to some previously described pauses (Kireeva and Kashlev, 2009), the pause at position 15 on the ST sequence was independent of the identity of dNMP in position 16 of the template (Supplementary Figure 3A).
We measured the proportion of RNAPs that pause at position 15 of the ST template (EC15ST) before being extended further.
The pause at position 15 thus may be a first experimental evidence for a factor-independent translocation pause, that is, of an 'in pathway' pause.
Stabilization of EC15ST in the pre-translocated state suggests that the pause at position 15 is caused solely by slow translocation.
The PEx analysis of OL ends in RNase A-treated DNA samples revealed a strong pause at position 5762 in both control and patient fibroblasts, likely owing to a hairpin secondary structure at OL reported previously (30).
The ST sequence also caused a pause at position 16, which corresponds to the backstepped EC16ST, suggesting that recognition of the RNA DNA hybrid sequence during transcription may also stimulate backtracking and as a result pausing of transcription (see also below).
In contrast, reduction of the pause at position 22 of WT template may be due to thermodynamic effect of weakening of the rear edge of the RNA DNA hybrid (in the similar manner as weak base pairs in position −8 destabilize backstepped state in Figure 2B).
