Tuesday, September 16, 2008

How RNA Polymerase Binds to DNA

Most cells contain two forms of RNA polymerase. The "core" polymerase is the part that carries out transcription of a gene where the DNA sequence is copied to produce a single-stranded RNA molecule. The core polymerase binds DNA non-specifically as you might expect for a DNA binding protein that has to travel down a large number of different genes.

When transcription is terminated the core RNA polymerase is released. In order to start a new round of transcription, the core RNA polymerase has to be directed to bind at a promoter, defined as the specific DNA sequence where transcription is initiated. There are specific DNA binding factors that bind to promoters and to RNA polymerase. That's how they direct RNA polymerase to the place where transcription has to start. These factors bind first to core polymerase forming the second form of RNA polymerase called the holoenzyme.

The binding parameters of the E. coli core polymerase and the holoenzyme have been studied in detail. In E. coli cells there are several different versions of holoenzyme. Each one contains a different initiation factor that binds to a different series of promoters. The most common initiation factor is called σ70 (sigma-70) and it binds to most of the promoters in the cell.

The steps in transcription initiation are shown in the figure. First, holoenzyme consisting of core polymerase + σ70, binds non-specifically to any stretch of DNA. It then moves along the DNA in a one-dimensional search until it finds a promoter sequence. This is followed by a local unwinding of the DNA and synthesis of a short piece of DNA.

Subsequent steps (not shown) require the dissociation of the initiation factor (σ70) and the formation of an elongation complex. RNA polymerase is then free to leave the promoter region and move down the gene making RNA.

THEME:
Transcription

The same kinds of parameters that we discussed yesterday are used to describe RNA polymerase binding [see DNA Binding Proteins and Repression of the lac Operon]. The core RNA polymerase by itself binds DNA non-specifically with an association, or binding, constant (Ka) of 1010 M-1. This is very tight binding for a DNA binding protein. Once bound to DNA the core RNA polymerase dissociates very slowly (t1/2 = 60 minutes).

The holoenzyme can also bind non-specifically. In this case the association constant is 5 × 106 M-1 and the complex dissociates rapidly (t1/2 = 3 seconds). The holoenzyme binds specifically to promoter sequences with an association constant of 2 × 1011 M-1 and t1/2 = 2 to 3 hours. Thus, the interaction of the initiation factor with core RNA polymerase has two effects: it decreases the affinity for random stretches of DNA and increases the affinity for the promoter sequence.

A typical E. coli cell contains about 5000 molecules of RNA polymerase. When the cells are growing rapidly, 2500 molecules will be bound to genes in transcription complexes. Another 1250 will be in initiation complexes of various sorts and most of the remaining RNA polymerase molecules (1200) will be bound to DNA non-specifically. Only a small number (~50) will be free in the cytoplasm.

Since the holoenzyme molecules are capable of initiating transcription on their own, a small number of the non-specifically bound molecules will accidentally transcribe short stretches of DNA. These spurious transcripts don't usually cause a problem since they are quite rare. Nevertheless, their presence means that much of the intergenic DNA in the E. coli genome is transcribed at one time or another.

Eukaryotic cells contain three different kinds of RNA polymerases [Eukaryotic RNA Polymerases]. Each one is much more complex that the bacterial enzymes but the principles of transcription initiation are the same.

In eukaryotes there are about a dozen general initiation factors for each of the different RNA polymerases. The ones for RNA polymerase II—the enzyme that transcribes protein-encoding genes—are called transcription factor IID (TFIID) etc. All of the factors are required for specific RNA polymerase binding at a promoter site and all of them associate with the core RNA polymerase to form a large holoenzyme complex. The eukaryotic general initiation factors do the same thing for eukaryotic core polymerase as the bacterial ones do for the bacterial RNA polymerase; they convert the complex to a specific DNA binding protein and lower its affinity for binding non-specifically.

As is the case in bacteria, a substantial number of holoenzyme complexes will be bound non-specifically to DNA at any one time. The proportion is much, much higher in mammalian cells because of the presence of so much junk DNA in the genome. This has the effect of soaking up a lot of holoenzyme complexes.

Since the holenzyme complexes, like those in bacteria, are capable of initiating basal levels of transcription, we should not be surprised to find spurious transciption in all parts of the genome. These transcript will be rare but they will come from any site where RNA polymerase holoenzme can bind.


Bibliography


Bustamante,C., Guthold,M., Zhu,X., and Yang,G. (1999) Facilitated target location on DNA by individual Escherichia coli RNA polymerase molecules observed with the scanning force microscope operating in liquid. J. Biol. Chem. 274, 16665-16668.
Goodrich,J.A., Cutler,G., and Tjian,R. (1996) Contacts in context: promoter specificity and macromolecular interactions in transcription. Cell 84, 825-830.
Koleske,A.J. and Young,R.A. (1995) The RNA polymerase II holoenzyme and its implications for gene regulation. Trends Biochem. Sci. 20, 113-116.
Myer,V.E. and Young,R.A. (1998) RNA polymerase II holoenzymes and subcomplexes. J. Biol. Chem. 273, 27757-27760.
Ossipow,V., Tassan,J.P., Nigg,E.A., and Schibler,U. (1995) A mammalian RNA polymerase II holoenzyme containing all components required for promoter-specific transcription initiation. Cell 83, 137-146.
Pan,G., Aso,T., and Greenblatt,J. (1997) Interaction of elongation factors TFIIS and elongin A with a human RNA polymerase II holoenzyme capable of promoter-specific initiation and responsive to transcriptional activators. J. Biol. Chem. 272, 24563-24571.
Ricchetti,M., Metzger,W., and Heumann,H. (1988) One-Dimensional Diffusion of Escherichia-Coli Dna-Dependent Rna-Polymerase - A Mechanism to Facilitate Promoter Location. Proceedings of the National Academy of Sciences of the United States of America 85, 4610-4614.
Shimamoto,N. (1999) One-dimensional diffusion of proteins along DNA. Its biological and chemical significance revealed by single-molecule measurements. J. Biol. Chem. 274, 15293-15296.
Singer,P. and Wu,C.W. (1987) Promoter search by Escherichia coli RNA polymerase on a circular DNA template. J. Biol. Chem. 262, 14178-14189.
Singer,P.T. and Wu,C.W. (1988) Kinetics of promoter search by Escherichia coli RNA polymerase. Effects of monovalent and divalent cations and temperature. J. Biol. Chem. 263, 4208-4214.
von Hippel,P.H. (2007) From "simple" DNA-protein interactions to the macromolecular machines of gene expression. Annual Review of Biophysics and Biomolecular Structure 36, 79-105.
von Hippel,P.H. and Berg,O.G. (1989) Facilitated target location in biological systems. J. Biol. Chem. 264, 675-678.

4 comments:

  1. Thanks for the post! Things like this deserve their own Knol - especially since you are an authority on the subject.

    Any plans for similar discussions of RNA binding proteins (La, HuR, Puf, etc)?

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  2. These transcripts will be rare but they will come from any site where RNA polymerase holoenzme can bind.

    Exactly how rare is the key question. Since most of the human genome is repetitive, spurious transcription would predominantly produce RNA to these repetitive sequences. Presumably, there is no strand preference to the initiation of this spurious transcription, so (+) and (-) strands of repetitive regions will be produced with approximately equal frequency. These (+) and (-) repetitive sequence transcripts are complementary to each other and should be able to anneal to form double-stranded RNA to these repetitive regions. Is this dsRNA to human repetitive genomic elements detected?

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  3. Thanks Dr. Moran,

    I really appreciate these kinds of posts. I am a non-bioligist and while the material is a little over my head, I will resarch further on my own to answer any questions.

    I read your blog often.

    Dennis

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  4. Great post. Thanks for sharing.Klenow (3′→ 5′ exo-) is a mesophilic dna polymerase deficient in both proofreading (3′→ 5′) and nick-translation (5′→ 3′) nuclease activities, and that displays a moderate strand displacement activity during DNA synthesis.

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