RNA Polymerase

  T7 RNA Polymerase 

Introduction and Transcription

     RNA  polymerase is a protein crucial to the Central Dogma of biology.  The enzyme facilitates the cellular process of transcription and without it, no new RNA or proteins would be produced.  The actual protein is composed of a core polymerase and a sigma factor, which together make up the holoenzyme.  The catalytically-active core polymerase has five subunits, however, it is not able to recognize DNA on its own.^[1]  The sigma factor recognizes the DNA and binds to the transcription initiation sites, or cis-acting elements, on DNA (Berg, Jeremy et al. 2002).  The figure below shows the holoenzyme as well as the -35 and -10 promoters where polymerase binds.

This figure shows a general promoter and where the RNA Polymerase sigma factor binds.^[2]

     Transcription is the cellular process of producing RNA.  The main steps involved are the melting open of double-stranded DNA, writing RNA off of a DNA template, and releasing newly-formed RNA, each of which are facilitated by RNA Polymerase.  The most important process involved in transcription is forming phosphodiester bonds between each new nucleotide.  This thermodynamically-favored process involves the addition of a new nucleotide to the 3' OH group onto the growing RNA strand (Berg, Jeremy et al. 2002).

For a detailed animation of how RNA Polymerase functions in transcription, click here.

Function of RNA Polymerase

     RNA polymerase is the most crucial enzyme in transcription and serves as an endpoint in the various pathways that regulate the expression of certain genes.^[3] 

     -The first steps in transcription are binding and initiation.  RNA polymerase searches the DNA strand for the correct initiator and then binds to the promoter to form a closed complex (Griffiths, Anthony et al. 1999).  The polymerase then unwinds the double-stranded DNA (about 14 base pairs) to form the open complex which allows the RNA polymerase to read the single strand of DNA.^[4]  The melting open of the dsDNA is one of the two rate limiting steps of transcription because it happens very slowly (Madame Curie Bioscience Database). The initiation site is where new nucleotides are first added within the open complex.^[5]  RNA polymerase stays on the DNA strand and chain elongation occurs. 

     -Elongation, or the synthesis of RNA, occurs until the polymerase reaches the terminator signal.  RNA polymerase continues along the DNA strand, unwinding DNA and transcribing RNA as it moves (Cordts, M. and Merkel, S. 2002).  The transcription bubble is always present on the DNA strand, but continues to move with the polymerase.

    -Termination, or the release of the newly-formed RNA strand, occurs through two basic mechanisms:  rho dependent or rho independent.  In rho dependent termination, the rho protein (which is similar to helicase) slides along the RNA strand and releases it from the RNA polymerase (Transcriptional Termination in Prokaryotes).  Rho independent termination occurs when a specific sequence on the DNA  causes the RNA strand to form a stem-loop structure followed by a series of Uracil nucleotides (Transcriptional Termination in Prokaryotes).  This series of Us causes the RNA to break off and be released from the polymerase. 

This figure shows the basic mechanism of RNA Polymerase during transcription.  An unwound piece of DNA is

always present and is about 10.5 base pairs in length.  This transcription bubble is where the template is being copied.^[6]

     There are many types of RNAs made from transcription, however, molecular biologists most often deal with mitochondrial RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA).

Crystal Structure

     The crystal structure of RNA polymerase shows us the secondary structure of the protein, including the α-helices and ß-pleated sheets, as well as the tertiary and quaternary structure.  The quaternary structure shows the different subunits of RNA polymerase and from it, we can get an idea about what sorts of purposes each subunit serves.  The crystal structure of the holoenzyme allows one to visualize the initiation of transcription as well as the open and closed complexes formed throughout transcription (Vassylyev, Dmitry et al. 2002).

RNA Polymerase and Disease

     There is not a specific human disease produced by mutations in RNA Polymerase, but mutations can lead to Rifampin resistance (Vattanaviboon, P et al. 1995).  Rifampin is an antibiotic which inhibits RNA synthesis by constricting the core polymerase's ß-subunit and stops transcription (Wikipedia).  It is used to treat Tuberculosis, however, the antibiotic is not used frequently because it is so susceptible to viral resistance.  Rifampin is very effective against Tuberculosis if the virus is not already resistant or immune (Rifampicin Complex).  Only three pharmaceutical companies still make Rifampin because TB is not a huge problem any more in the western world (Rifampicin Complex). 

Rifampin's structure (Wikipedia).

     A specific polymerase binding protein, RbpA, has also been shown to be partially resistant to Rifampin (Newell, Katy V. et al. 2006).  Since polymerase binding proteins interact with RNA polymerase, the two proteins together constitute antibiotic resistance. 

     Mutations in poliovirus RNA polymerase can also lead to resistance to vaccinations against the bacteria (Pfeiffer and Kirkegaard 2003).   

     RNA polymerase is also being investigated for use as a theraputic agent against Dengue fever.  The Dengue RNA polymerase non-structural protein 5 is the specific protein being studied.  Because there is no treatment for the Dengue fever infection, this research is quite promising and scientists are hopeful for its success (Rawlinson, Stephen M. et al. 2006).