Pol II and Friends

Finding Pol I, II, III 

When scientists found the eukaryotic RNA Polymerases I II, and III, they did so by separating the proteins by chromatography. A poison from a mushroom called a-amantin was passed with a protein extract from the nuclei of cultured eukaryotic cells through a DEAE Sephadex columun. After the protein was collected at various different times, it was eluted (washed) with a solution of constantly increasing NaCl. Fractions from this elute were assayed for RNA polymerase activity (is the RNA synthesized or not?), and it was found that where Pol I was insenitive to a-amantin at both 1 umg and and 10 umg, Pol II was sensitive to a-amantin at 1 umg, and Pol II was intermediately sensitive to a-amantin, meaning insensitive at 1 umg, but sensitive at a higher concentration of 10 umg.

Introduction to the Three Eukaryotic Polymerases

Polymerase	RNA Transcribed	RNA Function
Polymerase I	Pre r-RNA (28S, 18S, 5.8S rRNAs)	ribosome complements, protein synthesis
Polymerase II	mRNA snRNA siRNA miRNAs	encodes protein RNA splicing chromatin-mediated repression translational control translation control
Polymerase III	tRNA 5S rRNA snRNA U6 7S RNA other stable short RNAs	protein synthesis ribosome component, protein synthesis RNA splicing signal-recognition particle for insertion of polypeptides into the ER various functions, most unknown

Conservation in Polymerase Structures

It isn't too surprising that there are highly conserved structures between bacterial E coli RNA polymerase (bacteria) and Yeast RNA polymerase (eukaryotes). After all, transcribing RNA from DNA is pretty important to life, and conservation indicates the polymerase enzyme was around early on in its evolutionary history. So not only does yeast RNA polymerase share similar major structures similar to the B and B' E coli RNAs, but also possesses a w-like and two nonidentical a-like subunits.

However, the eukaryotic RNA polymerases are more complex than the bacterial counterparts. All three yeast RNA polymerases contain four additional subunits common to them, but not shared with prokaryotes.

Also each eukaryotic nuclear RNA polymerase has several enzyme specific subunits that are not present in the other two RNA polymerases. Three of these additional subunits of Pol I and Pol III  are homologous to three of the additional subunits of Pol II. The other two Pol I-specific subunits are homologous to Pol II transcription factor TFIIF. The four additional subunits of Pol III are homologous to the Pol II transcription factors TFIIF and TFIIE. So in conclusion, Pol II has three additional specific subunits, Pol I has 5, and Pol III has 7.

* Structure of Yeast RNA Polymerase
I may go back to this post and add more details about the Yeast RNA Polymerase domains, but as of now, I am unsure if this level of intricacy is required. If I do, you will see an additional paragraph section deal added tomorrow, if not, this little blurb will be deleted.

Carboxyl-Terminal Domain (CTD) 

This is the largest subunit on the RNA Polymerase II, which contributes to the vitality of the organism. Without a certain number of repeats (10 in yeast), the polymerase will not function and the organism will die. The stretch of 7 amino acid is nearly always precisely repeated, and is Tyr-Ser-Pro-Ser-Thr-Pro-Ser in mammals. A cute way to remember this sequence is the statement "this subject proves that some people study"; sorry it is kind of negative, but the best I could come up with under strain. Yeast had 26 or more of these repeats, where as vertebrates have 523.

RNA pol II molecules that first initiate transcription have an unphosphorylated tail, but once the polymerase initiates transcription and begins to move away from the promoter, many of the serine and some tryosine residues in the CTD are phosphorylated.

This phenomenon can be seen in vivo in Drosphilia staining. A puffed chromosomal region is stained by antibodies specific for phosphorylation (red) or unphosphorylated (green) from the salivary glands.

Now, as an aside, I've never been able to stain Drosphilia salivary glands successfully. I decapitated my larvae three or four times with painful lack of precision, and when asking a TA for assistance, was informed that no one knows how to make the puncture wound to spew out the desired contents under the dissection microscope. I might be just a little sore about this failing, as three to four Drosphilia died needlessly in infancy and did not make it to the next test cross where they eventually achieved the honour of death by morgue jar. Anyway, my lab partner was kind of squeamish about the process, and out of a class of thirty only one pair managed to find the classic five stranded chromosome. I digress. But anyone who can stain a chromosome with two types of antibodies, get the bally chromosome from larvae guts in the first place, and see the regions of interest, is in my opinion, kind of a boss. Kudos to them.