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Wednesday, March 05, 2014

The crystal structure of E. coli RNA polymearse σ70 holoenzyme

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The Journal of Biological Chemistry (JBC) publishes a little booklet of the "best of jbc." The latest copy arrived in the mail a few days ago and it alerted me to a paper published one year ago on the structure of Escherichia coli RNA polymerase σ70 holoenzyme (Murikami, 2013).1

The control of transcription initiation is a very important topic in biochemistry and molecular biology and the events in E. coli are the model for transcription initiation in all other species. We know more about RNA polymerase and promoter sites in E. coli than in any other species.

You can see how it works by reading: How RNA Polymerase Works: The Chemical Reaction , How RNA Polymerase Works: The Topology of the Reaction and the Structure of the Enzyme , and How RNA Polymerase Binds to DNA. Or you can read a good review by Murakami and Darst (2003).

Here's a brief refresher to bring you up to date. E. coli RNA polymerase is a large enzyme consisting of five subunits: two copies of the α subunit and three others (α2ββ′ω). There are many different transcription regulatory proteins in the cell and each one recognizes and binds to a different type of promoter. Most of them are called "sigma (σ) factors." The most common factor is σ70— it's the one that recognizes the traditional TATA box at the transcription initiation site of most E. coli genes.

The transcription activators bind to RNA polymerase to form a "holoenzyme" complex. This complex binds DNA and slides along it until it encounters the specific sequence recognized by the σ factor. This is the promoter region. The next step involves unwinding a bit of DNA to create a transcription bubble. Then the σ factor dissociates and transcription begins.

The new crystal structure shows the location of σ70 for the first time. It's a medium-sized protein (M.W. = 70,000) with five domains. The σ2, σ3, σ4 and σNCR domains form a tight interaction with core RNA polymerase but the flexible end of σ70 is the N-terminal domain (σ1.1) that binds weakly to the main groove where DNA interacts with the holoenzyme. This covers the active site of the enzyme where the first few nucleotides will be polymerized. That σ1.1 domain is shown as a yellow outline in the figure from the paper (below right). The structure of σ1.1 was difficult to resolve in the electron density map, indicating that it is not tightly bound.

The idea is that the exposed surface of the σ2 and σ3 domains bind to the TATA box and position the promoter DNA in the blue grove labelled "promoter DNA" in the lower image. (The blue surface indicates basically-charged regions that can bind to the negatively-charged DNA molecule.) Note that the orientation of the molecule in the figure from the paper is the reverse of the figure from my book where the promoter region is on the left.

The flexible σ1.1 domain prevents formation of the open complex unless the rest of the protein has found a true TATA box. When that happens, the σ1.1 domain probably uncovers the rest of the DNA grove exposing the site for binding "downstream DNA" and formation of the open complex.

Once the open complex forms, the entire σ factor can dissociate and transcription begins.

Into the textbook it goes!


JBC publishes a lot of papers and I don't have time to scan the table of contents every few weeks. It's easy to miss important papers this way. I rely on others to find them but that doesn't always work. This is a good example.

Murakami, K.S. (2013) X-ray Crystal Structure of Escherichia coli RNA Polymerase σ70 Holoenzyme. Journal of Biological Chemistry 288:9126-9134. [doi: 10.1074/jbc.M112.430900]

Murakami, K.S. and Darst, S.A. (2003) Bacterial RNA polymerases: the wholo story. Current Opinion in Structural Biology 13:31-39. [doi: 10.1016/S0959-440X(02)00005-2]

17 comments :

mregnor said...

Beautiful stuff and great science, Larry.

You think it all just happens by chance?

Diogenes said...

Smegnor, if you think this complex was formed by magic, how would you test that hypothesis?

This is an E. coli enzyme. Some E. coli are pathogenic. Are you saying your God designed pathogenic bacteria in order to make us sick? He would be a psychopath, no?

Smegnor, can you point to any human artifact which resembles this complex? Human machines are made from rigid parts or metal or wood that fit together perfectly. The business end of this protein is a functional, but floppy, barely structured N-terminal domain that wobbles around, and its main function is to unravel and become unstructured when other bits interact with negatively-charged DNA. Do humans ever design unstructured functional things? Do humans use "induced fit" in a machine where the bits don't fit together until the electrical charges of their partner make them flop around?

Do humans ever design "machines" with flexible, floppy bits whose function is to unravel and become unstructured?

If not humans, then do you have any evidence of spooks or spirits making such floppy bits, or for that matter, altering even one amino acid in any protein from the proteome of any species anywhere? Even one? One? Bueller?

John Harshman said...

Gosh, no. Larry thinks it was magically poofed into existence by an invisible, undetectable man in the sky. Doesn't everyone?

Diogenes said...

How the hell did this paper wind up in JBC? It's not that bad. Are we so bored with multi-protein complexes of great functional significance?

steve oberski said...

I'm quite sure the noodley appendage of the Flying Spaghetti Monster was involved in the design.

He would be a psychopath, no?

I've noticed that the gods that people (I'm using the term loosely in mregnor's case) pretend to believe in tend to act like those very same people.

Anonymous said...

JBC is one of the best serious scientific journals out there.

Devin said...

JBC is legit, man. But crystal structures need to be either extremely difficult or very high impact in order to get into something Nature or Science.

SPARC said...

I got to know the TA-rich element in prokaryotic promoters as the "Pribnow-box" rather than the "TATA-box". Although the later sequence confers similar functions in eukaryotes I doubt that the terms are considered interchangable.

Peter Perry said...

I think Diogenes confused the Journal of Biological Chemistry (a scientific journal) with the Journal of Biological Complexity (christian apologetics journal).

Newbie said...

Larry,

I just saw a guy that looked like you at the panda express in Miss! My wife pointed him out but I was not sure....

SRM said...

this paper follows a large number of structural papers for bacterial RNAP holoenzyme stretching back to the first determination by Murakami and Darst (2002).

You want to see some stunning structural work, check out this paper that shows two nucleotides from the non-template strand of promoter flipping up into pockets formed by sigma region 2 amino acids. This is what traps the promoter in a single-stranded state and allows transcription initiation.

http://www.ncbi.nlm.nih.gov/pubmed/22136875

SRM said...

actually, I thought the eukaryotic TATA box plays a role more akin to the -35 site of the bacterial promoter, not the -10 (pribnow) site.

Diogenes said...

Pedro says: "I think Diogenes confused the Journal of Biological Chemistry (a scientific journal) with the Journal of Biological Complexity"

The TOC of the Journal of Biological Chemistry makes my eyes glaze over. Reading JBC is like reading a phone book.

Diogenes said...

Your "wife", "LouiseG"?

"LouiseG" says: "I just saw a guy that looked like you at the panda express in Miss! My wife pointed him out but I was not sure.... "

Right. "Louise", we already knew you were a sock puppet for Quest, and as such you're banned.

Anonymous said...

My sock puppet..? Are you out of your mind...? Probably Witton or another banned iditot is trying to play a game with Larry and others...

I know you want me banned, so that you can spread your shit about how evolution is true even though nobody, including you, can agree on the mechanism of it....

I'm not even going to mention abiogenesis you atheistic and naive coward....

BTW: Joe Francs has tried the same move on me by claiming I was Witton....

I'm not even going to mention why....lol

SRM said...

actually I will take back that comment about TATA being akin to -35 except perhaps from a spatial perspective. In any case, the eukaryotic TATA box should not be considered very analogous to the bacterial -10 element (which for the primary sigma has the consensus sequence of TATAAT). In bacteria this region coincides with nucleation of melting whereas in eukaryotes melting occurs about 20 nt downstream of the TATA box.
I agree that the bacterial -10 element should not be called a TATA box although perhaps it has been in the past for obvious reasons.

Rolf Aalberg said...

I know you want me banned, so that you can spread your shit about how evolution is true even though nobody, including you, can agree on the mechanism of it....
You show us your mechanism first... Then you try to wrap your mind around the fact of natural selection. Then we take abiongenesis.... There is a time for everything and now is the time for creationists to take off their blinders - the problem is that they don't see them...