Pol II caught speeding by single gene imaging

EMBO Rep (2011) advance online publication. doi:; DOI: 10.1038/embor.2011.196

The transcription elongation rate of RNA polymerase II (Pol II) has been re-estimated persistently for more than 30 years using a variety of methods. A new imaging-based estimate of elongation rate on an HIV-derived transgene implies Pol II can elongate at least an order-of-magnitude faster than previously thought.

From early pulse-labelling experiments to recent studies monitoring post-stimulus waves of transcription using Pol II chromatin immunoprecipitation (ChIP), tiling arrays or reverse transcription (RT)–PCR, estimates of Pol II elongation rate from various eukaryotic cell types have ranged between 1 and 6 kb/min (Ardehali & Lis, 2009; Wada et al, 2009). Imaging approaches, using differences in hybridization of differently positioned RNA fluorescence in situ hybridization (FISH) probes, and more recently live-cell RNA detection, have generated elongation rates similar to those measured using test-tube processing methods. Quite unexpectedly, a study by Marcello and colleagues published in this issue of EMBO reports shows that the RNA Pol II elongation rate can be as fast as 100 kb/min (Maiuri et al, 2011).

Detecting RNA in living cells uses specific high affinity RNA–protein interactions, through which GFP can be directed to RNA, in living cells. The most commonly used technique uses RNA stem loops from the genome of the MS2 RNA bacteriophage (Bertrand et al, 1998). MS2 stem loops are included in the gene of interest, and upon transcription are incorporated into RNA, where they are detected using a fusion of GFP to the MS2 coat protein, which has a high-affinity, sequence-specific interaction with the stem loops. If strongly transcribed, nascent RNA can be detected at the transcription site as a fluorescent spot. To measure transcription rates, recovery of the spot fluorescence is monitored after photobleaching. The stem loop–MS2 coat protein interaction is stable (Boireau et al, 2007), so fluorescence recovery is determined by the synthesis rate of new stem loops.

…Pol II can elongate at least an order-of-magnitude faster than previously thought

Initial bleaching-based estimates of the Pol II elongation rate have used multicopy insertions of vectors expressing MS2 loops from heterologous promoters. Estimates (see Table 1) range from 1 to 4.3 kb/min (Ben-Ari et al, 2010; Boireau et al, 2007; Darzacq et al, 2007). The advantage of multicopy arrays is a high signal-to-noise ratio, with most signal due to bound MS2–GFP, rather than freely diffusing molecules. Furthermore, the system can be considered to be steady state, greatly facilitating analysis. However, use of tandem arrays overlooks the precise dynamics of individual transcription sites, which might have multiple states and multiple rates (Chubb & Liverpool, 2010; Raj & van Oudenaarden, 2008). It is also unclear to what extent multicopy insertions, which can be several megabases of heterologous sequence, behave as native genes. As RNA detection moves towards single gene approaches, so reducing the impact of these issues, surprising observations about polymerase elongation rates are emerging.

Table 1. Comparing measurements of Pol II elongation rates.

System	Number of arrays	Promoter	Elongation rate (kb/min)	Reference
Various non-imaging	N/A	N/A	1–6	Ardehali & Lis, 2009
U2OS	200	Modified CMV	4.3	Darzacq et al, 2007
U2OS	70–75	HIV LTR	1.9	Boireau et al, 2007
U2OS	6	Modified CMV	3.3	Ben-Ari et al, 2010
Yeast Pol I gene	1	Endogenous	1.2 or 2.76	Larson et al, 2011
HOS_A4	35	HIV LTR	1	Maiuri et al, 2011
HOS_A4	1	HIV LTR	50–100	Maiuri et al, 2011
HEK-293	1	CMV or CCND1	0.31–0.78	Yunger et al, 2010

N/A, not applicable.

Using single integrations of HIV-based vectors, with MS2 transcription driven by the long terminal repeat (LTR) promoter, Marcello and colleagues estimated Pol II elongation rates of 50–100 kb/min. After photobleaching, RNA spots quickly recovered, reaching equilibrium in less than 10 s, contrasting the 300 s required for recovery of some multicopy arrays. By using bleach recovery times and knowledge of the gene length, an elongation rate of 80 kb/min was estimated. The rate was re-evaluated using a computational model that considered peaks of a harmonic-positive travelling wave to represent each polymerase moving constantly along the gene. This method determined the elongation rate to be above 50 kb/min and approaching 100 kb/min, in line with the initial estimate (Maiuri et al, 2011). In the same study, Marcello and colleagues measured the elongation rate of the same vector integrated in the genome as 35 tandem repeats. The bleached spot recovered tenfold slower than a single insertion, and the elongation rate (model-derived) was estimated at 1.6 kb/min, similar to earlier estimates from multicopy HIV-derived vector insertions (Boireau et al, 2007).

By using single integrations of HIV-based vectors, […] Marcello and colleagues estimated Pol II elongation rates of 50–100 kb/min

These observations raise several questions and underscore concerns over the use of large arrays. What is the chromatin environment at the single-copy transgene? What features of the enzyme and its associated factors are associated with fast transcription? Why do elongation rates depend so strongly on the number of tandem arrays of MS2 stem loops? A clue might exist in the differences between measurements of RNA load, which showed a more than tenfold increase in RNA per gene at the single locus compared with the multicopy insertion. Early observations of the properties of multicopy transgenes implied they can be heterochromatic (Janicki et al, 2004), potentially retarding access to the locus and impeding release of the transcript after synthesis, perhaps causing a block in transcription site dynamics. These issues are potentially magnified by non-native characteristics of the vector. The Marcello lab addressed the diffusion issue by bleaching free GFP in the region of the arrays and found no impediment to GFP diffusion, although GFP diffusion will be exposed to different constraints than will the diffusion of ribonucleoprotein particles, and diffusion measurements are not directly reflective of the establishment of transcriptional complexes.

…can we really expect a single elongation rate to be sufficiently descriptive, even for a single gene?

Although we should not dismiss effects of multicopy arrays on transcription kinetics, other single insertion measurements give different elongation rates to HIV transcription. Using MS2 transgenes integrated as single copies into heterologous sites in human HEK-293 cells, Shav-Tal and colleagues estimated Pol II elongation rates of 0.31–0.78 kb/min, below the ‘standard’ range (Yunger et al, 2010). A second single-copy study, based upon insertion of stem loops into an endogenous yeast gene, measured rates of 1.2 kb/min (early in the cell cycle) to 2.76 kb/min (late in the cycle; Larson et al, 2011). A feature of the yeast work is that estimates were not made from bleaching, but derived from measurements of fluctuations in spot intensity.

It remains to be seen whether the impact of the HIV LTR promoter on Pol II elongation is solely an unpleasant feature of viral transcription, or is reflective of a much broader general range of elongation rates than has been previously observed. A continued exploration of single gene dynamics will be necessary to address this further, and promises yet more complexities, as the analysis for most genes will not allow consideration of a ‘steady state’. Given the number of components involved in the regulation of transcription, can we really expect a single elongation rate to be sufficiently descriptive, even for a single gene?