RNA Polymerase Structure and Function

Yi Shu

Department of Biomedical Engineering

 


Background

Question

Hypothesis

Experimental Design

Useful Links
Background

RNA polymerase in bacteria

In bacteria, RNAP is a relatively large molecule. The core enzyme has 5 subunits α2ββ'σω (~480 kDa):

  • α2: the two α subunits assemble the enzyme and recognize regulatory factors.
  • β: subunit has the polymerase activity (catalyzes the synthesis of RNA) which includes chain initiation and elongation.
  • β': binds to DNA (nonspecifically).
  • ω: restores denatured RNA polymerase to its functional form in vitro.
  • σ: In order to bind promoter-specific regions, the core enzyme requires this subunit.

RNA polymerase in eukaryotes

Eukaryotes have several types of RNAP, characterized by the type of RNA they synthesize:

  • RNA polymerase I synthesizes a pre-rRNA 45S, which matures into 28S, 18S and 5.8S rRNAs which will form the major RNA sections of the ribosome.
  • RNA polymerase II synthesizes precursors of mRNAs and most snRNA and microRNAs.
  • RNA polymerase III synthesizes tRNAs, rRNA 5S and other small RNAs found in the nucleus and cytosol.
  • RNA polymerase IV synthesizes siRNA in plants.

RNA polymerase in viruses

  • Many viruses also encode for RNAP.
  • The most widely studied viral RNAP is found in bacteriophage T7. This single-subunit RNAP is related to that found in mitochondria and chloroplasts.
  • Shares considerable homology to DNA polymerase I family.

 Most information above comes from http://en.wikipedia.org/wiki/RNA_polymerase

 

T7 RNA polymerase is a good model to study RNAP function:

  • 98 kDa. it is smaller than the multi-subunit prokaryotic and eukaryotic RNA polymerases, facilitating X-ray crystallographic studies.

  • Homology between T7 RNA polymerase and the DNA polymerase I family is important for studying the functional differences between RNA and DNA polymerases.

  • The polymerase domain has a U-shape fold and  contains  the thumb, palm and fingers subdomains. The fingers sub-domain rises above the palm sub-domain forming a wall on the left of the active-site cleft and includes residues 554–784. Crystal structures of RNA polymerases demonstrate that the fingers subdomain interacts with both the DNA template  as well as nucleotides.

  • Y639 located in fingers sub-domain

 

 

Fig. 1 A. A structural comparison of the fingers sub-domains of RNAP and the KF fragment. Regions that superimpose are colored yellow and identical residues are shown in red. B. Primary sequence alignment of the fingers sub-domains of RNAP and the KF, based upon the structural super-position in (A). D.Jeruzalmi and T.A.Steitz. Structure of the T7 RNA polymerase–T7 lysozyme complex. The EMBO Journal Vol.17 No.14 pp.4101–4113, 1998.

Fig. 2   T7 RNAP structure. D.Jeruzalmi and T.A.Steitz. Structure of the T7 RNA polymerase–T7 lysozyme complex. The EMBO Journal Vol.17 No.14 pp.4101–4113, 1998.

Question

1. T7 RNA polymerase shares considerable homology to DNA polymerase I family. But why DNAP has high affinity of recognizing and binding of  2'-deoxynucleotides but RNAP have lower binding activity?

2. How does T7 RNAP distinguish ribo- and 2'-deoxynucleotides?

2. Previous study showed that Y639F mutant T7 RNAP had higher 2'-deoxynucleotides binding affinity than the widetype (wt) T7 RNAP, so why the mutant T7 RNAP acts as a DNAP?

 

Hypothesis

The Y639 is crucial for RNAP to distinguish ribo- and 2'-deoxynucleotides. The possible reason for the loss of the ribonucleotide discrimination of mutant T7 RNAP may be the loss of H-bond formation potential between Y639 and nucleotides 2'-group.

 

Experimental Design

Y839F mutant T7 RNAP

Method 1: Using modified nuleotides with different potential to form H-bond between 2'-group and Y639 to test the binding affinity of nucleotides and RNAP before and after Y639F mutation.

1. Nucleotides with different 2'-group (32P-labeled)

2. Binding test and kd value determination between different 2'-group nuleotides and Y639 RNAP.

3, Binding test and kd value determination between different 2'-group nuleotides and F639 mutant RNAP.

Method 2: Test the binding affinity between four kinds of nucleotides and RNAP before and after Y639H mutation (suppose Y639F mutation does not change the RNAP structure and acivity to bind ribonuleotides).

1. Y639H mutation

2. Four kinds of nucleotides

3, Binding test and kd value determination between different 2'-group nuleotides and H639 mutant RNAP.

We can draw the conclusion if:

1. The binding affinity of the four kinds of nucleotides with Y639 RNAP is direct propotion of their H-bond formation ability.

2. The binding affinity of the 2'-deoxynuleotides with F639 mutant RNAP is increased significantly,

3. The binding affinity of the four kinds of nucleotides with H639 mutant RNAP is direct propotion of their H-bond formation ability.

 

Useful links

1. Pubmed: http://www.ncbi.nlm.nih.gov/pubmed/

2: PBD database: http://www.rcsb.org/pdb/home/home.do