stevens at rockvax.rockefeller.edu
Thu Jun 15 08:55:53 EST 1995
daceh at shrike.und.ac.za (Halford Dace) wrote:
>In the absence of a seemingly appropriate newsgroup I thought I'd post
>this question here.
>The paper `A TBP-TAF complex required for transcription of human
>snRNA genes by RNA polymerases II and III' by R. William Henry et al.
>in Nature 374, 13 April 1995 refers.
>The above paper is the focus of a piece of work my class is doing in which
>we are asked (amongst other things) what `cryptic promotors' and `basal
>promotor elements' are. The context is roughly this:
>The authors isolated a new regulatory complex required for the transcription
>of snRNA genes. Its affinity for PSE (the basal promotor element in question)
>was assessed by various means, and the neccessity of the complex for
>normal expression of the U1 snRNA was also checked. Here's the rub:
>A gel (fig 1c for those who have the paper) is shown of the products of
>an in vitro transcription assay of SNAPc depleted nuclear extracts
>complemented with various density gradient fractions, a few of which
>contain the regulatory complex in question. Of course, only those
>show a band corresponding to U1 transcription, but all lanes show another
>band attributed by the authors to `RNAs initiated at cryptic promoters
>within vector sequences'.
>Could anyone clarify what this means? My guess is that it implies
>the presence of sequences within the cloning vector which act as
>promotors without them necessarily being intended as such - so nonspecific
>transcription just happens with or without the regulatory complex.
>Would I be correct here? Any helpful responses will of course be
>appropriately referenced when the work is handed in.
Since this project has been worked upon in our lab, and I have been writing a fellowship regarding it, I believe I can answer your questions in detail about snRNA transcription, however, general answers will perhaps suffice for what you are asking here.
First, it is important to know that if you give an RNA polymerase DNA and nucleotides and nothing else, you will get transcripts. In fact, this is an assay employed in isolating the enzyme. These transcripts are nonspecific and usually not at all significant.
Next, as nuclear extract is fractionated, there are a great many transcription factors that can be utilized showing up all over the place. In this case, it may be relevant that TBP, depending on the amount of extract loaded and reagents employed, can show up in almost every fraction. Furthermore, TBP can sometimes 'fall out' of specific complexes that might not give pol II specific transcription (ie SL1 or TFIIIB) and give activity in a fraction it would not be expected to exist in.
Just as there are a number of protein fractions that may support transcription, there are also a number of DNA sequences that may act as promoters. I have seen examples of seemingly-specific transcripts originating in 'random' DNA that upon further examination bear sequences that conform to canonical TATA or initiator sequences. The next obvious question is if these sequences show up everywhere, why don't we see transcripts all over the place in vivo? The reasons for this are probably more than I could list here, however there are some more important ones; in vitro transcripton is carried out with fractions and proteins many hundreds of times more pure and perhaps more concentrated (depending on how you define that in vivo) than in the nucleus, so you are stacking the deck in your favor. Additionally, the amount of core promoter transcription observed in vitro is a relatively small effect; we are just using a very sensitive detection system to pick it up. Finally, the nuclear DNA is packaged in such a way that core promoter transcription is typically absent. This nonspecific repression makes sense in terms of regulating the expression of critical genes-you would probably not want even low levels of expression of proto oncogenes at innapropriate times, thus you depend upon upstream activators for actually turning off and on the promoter.
The final answer is that sequences within the DNA can act as promoters because they look like genuine promoters, and there are most likely other members of the pol II general factors around to allow transcription to occur. Keep in mind just how pure these fractions are when wading through the data.
Our own work also addresses these issues-we are actually in competition with the lab in the paper you mention, so I guess I should tell you to read ours ;) ! Actually, if you would like to have a more detailed discussion, please e-mail me below.
Being a postdoc in a transcription lab, and finding little or no discussion regarding this topic, is there suffcient interest to think about a separate group? I have been thinking about it, but do not ahve the time to try to set it up, but would certainly try to contribute if someone else does! Any and all transcription questions or discussions are welcome by e-mail-I'd like to hear what people are doing.
Sean Stevens R.G. Roeder Laboratory The Rockefeller University
stevens at rockvax.rockefeller.edu
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