Biochemistry 3107 - Fall 2002

Bacterial RNA Polymerase

The first nucleic acid synthesizing enzyme

In 1955, Marianne Grunberg-Manago and Severo Ochoa reported the isolation of an enzyme that catalyzed the synthesis of RNA. For this work, Ochoa shared the 1959 Nobel Prize in Medicine with Arthur Kornberg (who received the Prize for his work on DNA polymerase I).
	Marianne Grunberg-Manago	Severo Ochoa

 

This enzyme could convert ribonucleoside diphosphates into RNA:

(NMP)_n + NDP --> (NMP)_n+1 + P_i

 

However, the enzyme had a number of unsettling properties. It did not need a template; and, it could use as little as 1 NDP or as many as 4 NDPs as substrate. In fact, the sequence of the product RNA depended entirely on the number and concentration of substrate NDPs.

These are not the properties of an enzyme that must faithfully copy the genetic material for expression!

We now know that Grunberg-Manago and Ochoa had isolated the enzyme polynucleotide phosphorylase which usually catalyzes the breakdown of RNA - not its synthesis!

The real E. coli RNA Polymerase

In 1960, the true enzyme was identified by 4 separate groups: Sam Weiss at the University of Chicago, Jerard Hurwitz, A. Stevens and J. Bonner. This enzyme required a template, used all four rNTPs as substrates and synthesized a product with a composition similar to that of the template, and it required Mg⁺⁺.

[25-rxn]

 

DNA-directed RNA polymerase catalyzes the synthesis of RNA on a DNA template following similar nucleophilic attack chemistry as DNA polymerase.

For a long time, E. coli was thought to contain just a single RNA polymerase. We now know that, although it has one major or principal RNA polymerase, it also contains (at least) six minor forms that are required or are used in special circumstances.

As with DNA polymerase III, there are two forms of RNA polymerase:

Core Enzyme

The core enzyme has four polypeptide subunits: alpha (a), beta (b), beta' (b'), and omega (w) in the stoichiometry a₂bb'w. The omega subunit was for many years considered a curiosity since no function could be ascribed to it and it did not appear to necessary to function. However, it is now known that omega is necessary to restore denatured RNA polymerase in vitro to its fully functional form. It may function by binding simultaneously to the N-terminus and C-terminus of the b' subunit. The omega subunit is a part of the Thermus aquaticus enzyme whose structure was determined in May 2002.

Core RNA polymerase can bind to DNA and catalyze the synthesis of RNA but it has no specificity. This form of the enzyme will bind (quite well, really) to non-specific DNA but it cannot recognize promoters.

 

 

Structure of Core RNA polymerase - from Seth Darst's laboratory at Rockefeller University	Structure of RNA polymerase holoenzyme - from Seth Darst's laboratory at Rockefeller University

 

Holo Enzyme

The RNA polymerase holoenzyme contains an additional subunit - sigma (s). The sigma subunit does two things:

 

• It reduces the affinity of the enzyme for non-specific DNA.
• It greatly increases the affinity of the enzyme for promoters.

 

The following table summarizes the subunits of E. coli RNA polymerase and their properties:

subunit															size aa	size (Kd)															gene										function
alpha (b)															329	36511															rpoA										required for assembly of the enzyme; interacts with some regulatory proteins; also involved in catalysis
beta (b)															1342	150616															rpoB										involved in catalysis: chain initiation and elongation
beta' (b')															1407	155159															rpoC										binds to the DNA template
sigma (s)															613	70263															rpoD										directs enzyme to the promoter
omega (w)															91	10237															rpoZ										required to restore denatured RNA polymerase in vitro to its fully functional form
Note:Links in the subunit column will take you to the SWISS-PROT database entry on the protein; Links in the gene column will take you to the EMBL database entry on the gene.

The subunits of RNA polymerase assemble into a structure that has the same "hand" like structure as DNA polymerases.

[25-2a][25-2b]

The structure of the Thermus aquaticus RNA polymerase holoenzyme has recently been solved to 4 Å resolution. It is clear that the sigma subunit is intimately associated with the enzyme and literally "buries" into the interior of the complex.

FROM: Katsuhiko S. Murakami, Shoko Masuda & Seth Darst (2002) Structural basis of Transcription Initiation: RNA Polymerase Holoenzyme at 4Å Resolution. Science. 296(5571): 1280-1284.

 

The sigma subunit

Sigma is a specificity factor. It directs RNA polymerase to the promoter and ensures that transcription is initiated only where it is supposed to be initiated.

The very fact that RNA polymerase depends upon a specificity factor to direct RNA polymerase to the correct promoter immediately offers a mechanism for controlling transcription. If different sigma factors directed RNA polymerase to different promoters, we could regulate the expression of different genes. The only problem then is one of regulating the different sigma factors so that their activity does not conflict with one another in the cell.

The principal sigma factor in E. coli is s⁷⁰ - so called because the protein is 70 kD in size. The corresponding holoenzyme containing this sigma factor is sometimes abbreviated: E·s⁷⁰.

E. coli also has six alternative sigma factors that are used in special circumstances:

 

s³² is required for the expression of heat-shock genes, i.e. genes that are only expressed when the cell is exposed to a high temperature and it must make special proteins in order to survive. Some parts of the sequence of s³² can be recognized in other bacterial sigma factors.

s⁵⁴ is present all the time and it is required for the expression of many genes that are involved for nitrogen metabolism. s⁵⁴ has a very different sequence and structure than the other sigma factors in E. coli and most bacteria; thus there are at least two different families of sigma factor proteins in bacteria.

The importance of s^S has been increasingly recognised in recent years. This sigma factor appears only as cells enter the stationary phase of growth. It is responsible for transcription of all of the genes whose products are required during stationary phase.

 

FROM: Ishihama, A. (2000) Functional Modulation of Escherichia coli RNA Polymerase. Annu. Rev. Microbiol. 54:499-518.
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The best known use of multiple sigma factors occurs in Bacillus subtilis. This bacterium will sporulate when it runs out of nutrients. The sporulation process requires an ordered sequence of gene expression which is regulated and timed by means of new sigma factors. One of the sigma factors expressed in the late stages of development is found only in the mother cell and only after a site-specific DNA rearrangement takes place (See Rearrangement of a Bacillus subtilis sigma gene during sporulation).

The following table lists some of the sigma factors found in Bacillus subtilis:

 

 

The program of gene expression during sporulation requires, in order of appearance: s^A as well as s^H, s^F, s^E, s^G and s^K. s^G is expressed and used only inside the developing spore.

s^K is expressed and used only in the mother cell that surrounds the spore. However, the gene for s^K is in two pieces. During sporulation, the two parts of the gene are joined by a site-specific recombination reaction -- but, as a consequence of the recombination, the DNA between the two parts of the gene is deleted!

This is OK for the cell! The mother cell is fated to die - its genetic content is no longer necessary - and it does not matter if it suffers an irreversible DNA rearrangement before it does so. The true genetic complement of Bacillus subtilis is still preserved, intact, within the spore.

 

Anti-sigma factors

The importance of anti-sigma factors has been established in recent years. These factors form complexes with their cognate sigma factor, thereby inhibiting its function. One example is FlgM, which is an anti-sigma factor for the flagellar sigma factor s^F. Another example is Rsd, which is an anti-s⁷⁰ factor. It is not present in exponentially growing E. coli cells. However, when E. coli enters stationary phase, Rsd is synthesized and acts to block the activity of s⁷⁰ thereby allowing s^S to associate with the core RNA polymerase and direct expression of stationary phase genes.

Control of sporulation in Bacillus subtilis also involves anti-sigma factors and anti-anti-sigma factors!

 

FROM: Hughes, K.T. and Mathee, K. (1998) The Anti-Sigma Factors. Annu. Rev. Microbiol. 52:231-286.

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