Structural Biochemistry/Nucleic Acid/RNA/RNA Polymerase

RNA polymerase is an enzyme that produces RNA and catalyzes the initiation and elongation of RNA chains from a DNA template. RNA is created using a process known as transcription. The RNA polymerase is a key component to this process. The reaction that this enzyme catalyzes for is: (RNA)n + Ribonucleoside Triphosphate ->/<- (RNA)n+1 +PPi. RNA polymerases are relatively large. The size of RNA polymerase in a typical eukaryotic cell is roughly 500kDa. In bacteria it is roughly 400kDa and in T7 bacteriophage it is roughly 100kDa. Their speed of transcription is about 50 bases per second. A typical mRNA that codes for an average protein takes about 20 seconds in a prokaryotic cell and about 3 minutes in a eukaryotic cell. It is primarily longer in eukaryotes due to the fact that eukaryotic genes contain many segments that contain introns.

Requirements to Function
For DNA polymerases to properly carry out their function they must have the following components present for catalysis to occur. 1.	A template of DNA. The preferable template is a double stranded DNA. Single stranded DNA may also work as a template but RNA strands or DNA-RNA hybrids may not be used. 2.	Activated precursors. The reactions require ribonucleoside triphosphates: ATP (Adenine -ribose-triphosphate), GTP (Guanine-ribose-triphosphate), ATP (Adenosine-triphosphate), and UTP (Uracil-ribose-triphosphate). Nucleotides with three phosphates to the 5’ carbon of the ribose sugar. 3.	Divalent metal. Unlike DNA polymerase, a primer is not needed but a divalent metal ion like magnesium ion or manganese ion is effective.

The direction of synthesis is from 5' to 3' and the synthesis is driven by the hydrolysis of pyrophosphate. There have been hybridization experiments that show RNA synthesized by RNA polymerase is complementary to its DNA template.



RNA Biogenesis Pol I, Pol II, and Pol III
Gene transcription takes place in the nucleus of eukaryotic cells and transcription is performed by three different multisubunit RNA polymerases, Pol I, Pol II, and Pol III. Still little is known today about the biogenesis of these RNA polymerases: from their origin of synthesis, the cytoplasm, to their arrival in the nucleus for transcription. Only until recently have studies shown that polymerase assembly intermediates, assembly factors and factors required for polymerase nuclear import exist in the cell cytoplasm. Pol II is the most identifiable one so is the basis of most studies on the biogenesis of RNA polymerase.

Structure and Assembly of RNA Polymerase II


RNA Pol II transcribes mRNAs and small non-coding RNAs and contains 12 polypeptide subunits. Each RNA Pol has their own specified role in RNA polymerase. They all have ten identical subunit catalytic cores. The peripheral subunits are what differentiate their structure and function; RNA Pol II has been determined to contain cores that allow it to model the homologous cores in Pol I and Pol III. Pol I and Pol III will bind to opposite sides of Pol II (binding to Rpb1 and Rpb2) and are then divided into three interacting subunits.



The assembly of eukaryotic RNA core was first identified in studies of bacterial RNA polymerase because RNA Pol II core subunits are exactly identical to that of bacteria. Assembly of RNA Pol II is initiated by the formation of the αα dimer which interacts with the β and forms a bound complex intermediate. The active cleft in the RNA Pol II is composed of β subunits which are formed in the final step of assembly, so the polymerase will not be catalytically active until it is complete. RNA Pol II in both bacteria and eukaryotic cells has both exhibited formation in equivalent manner.

Assembly in vitro experiments have also been conducted to determine the origins of RNA Pol II. Using three mutant large subunits, their assembly was followed with the use of pulse chase experiments. Scientists found that Rpb3 and Rpb3 were the first to interact, and the bound complex then interacts with Rpb1. However, because larger mutated subunits were used, final assembly could not be complete without the use of Rpb6, Rpb10, and Rpb12, which are not normally part of final assembly in normal sized RNA Pol II. RNA Nuclear Import

If any RNA subunits are lost during its assembly, there will be an excess of Rpb1 present in the cytoplasm, meaning that the polymerase needs to be fully assembled before it is allowed to enter the nucleus and take place in transcriptase. Pol II nuclear localization factors have been identified to be functional polymerase-interacting proteins in the cell. The accumulation of Rpb1 is caused by the depletion of GPN1 and GPN3. The expression of GPN1 will lead to the depletion of excess Rpb1. GPN1 binding to Pol II can also be directly influence the ability of GTP to bind properly. Homologs of GPN1 also aid in the biogenesis and final assembly of Pol II. GPN1 interacts with the CCT complex, which chaperones many subunits in the formation of Pol II.

Nuclear Import Signal
The components of Pol II, the subunits and GPN proteins, are unable to produce a nuclear import signal, therefore, which is why a Pol II cannot enter the nucleus until it is fully assembled, so it can produce a signal. Iwr1 is a factor that interacts with fully assembled Pol II and adapts a nuclear signal onto it. And deletion of Iwr1 leads to a accumulation of all the Pol II subunits, showing that lwr1 is most likely the key to proper final assembly. Iwr1 binds to the active site on Pol II and can “sense” completion by interacting with the Rpb1 and Rpb2 subunits, ensuring that Pol II is fully assembled; this acts as the final checkpoint before entering the nucleus. Because deletion of Iwr1 affects the concentration of subunits in the cytoplasm, a nuclear export signal is used to trigger the recycling of Iwr1. Currently Iwr1 is only know to effect the subunits and factors involving Pol II upon depletion, nothing has been found on how it affects Pol I and Pol II.

Biogenesis for RNA Pol I and Pol III
The origins of Pol I and Pol III may depend on the chaperones Hsp90 and R2TP because the client proteins for these two chaperones were discovered to be the subunits of Pol I and Pol III. This makes sense because the deletion of A135, the Pol I subunit, results in Hsp90 binding to Pol I’s larging subunit, A190. Several bleaching experiments have been conducted on Pol I that revealed Pol I is assembled at the promoter sites. It unclear as to what happens to Pol I after transcriptase because it remains as a stable complex and does not dissociate, scientists are trying to determine whether or not Pol I is fundamentally different in other organisms.

Pol III is the least understood polymerase out of the three. A NLS sequence was discovered near the N-terminus of the second larger Pol III subunit, C128, and when this sequence is deleted it leads to the accumulation of C128 in the cytoplasm and other Pol III subunits. However, the other Pol III subunits remained intact and nuclear. This reveals that the core of Pol III is assembled within the cytoplasm and the released subunits bind the core of the nucleus. It appears that Pol III follows the same assembly pathway as that of Pol II, as revealed by native mass spectroscopy.

Due to the fact that all three RNA polymerases have at least ten identical subunits, we can draw the conclusion that all three polymerases can coordinate and simultaneous assembly. The study of a certain subunit in any of three polymerases can be better understood by also studying the other subunits at that stage of biogenesis.

RNA Polymerase Translocates
RNA molecules thousands of nucleotides long are synthesized by multi-subunit DNA-dependent RNA polymerases. Nucleotide condensation’s reiterative reaction happens at rates of tens of nucleotides per second. This is consistently linked to the translocation of the enzyme along the DNA template (threading of the DNA and emerging RNA molecule through the enzyme. This reiteration of the nucleotide addition/translocation cycle without separating the DNA from the RNA involves both isomorphic and metamorphic conformational flexibility to such a magnitude that it accommodates the essential molecular motions.

Eukaryotes
Eukaryotic cells have three types of RNA polymerases. Pol I: This type of RNA polymerase synthesizes RNA for the large subunits of ribosomes. Ribosomes are pretty much the protein making organelle in cells. Pol II: Creates mRNAs. Messenger RNAs provide a template for protein synthesis for ribosomes. It also creates many small nuclear RNAs which help modify RNA after they are formed. Pol III: Creates tRNAs. Transfer RNAs is basically for the small subunit of ribosomes.

These three types of polymerases can be distinguished from one another in lab by the level of inhibition by the alpha-amanitin poison. PolI is completely resistance to this poison. PolII is highly sensitive to this poison. And PolIII is moderately sensitive.

RNA polymerases in eukaryotic cells are composed of several subunits. Majority of them are small and unique to each type of polymerase. However there are two large subunits that are similar among all of the polymerases. This fact highlights that all these polymerases must have evolved from an original polymerase. The two large subunits are the functional core of this enzyme. The other smaller subunits tend to provide the specific functions for each distinct type of polymerase.

Bacteria
In bacteria, the RNA polymerase holoenzyme is made up of two parts, a core polymerase and a sigma factor. The core polymerase has the components needed for elongation in transcription, while the sigma factor is only needed for transcriptional initiation. The core polymerase is made up of two α’s, one β, and one β’ unit (α2 β β’), while the sigma factor is only made up of s. In total, there are 5 subunits in RNA polymerase-- alpha (α), beta (β), beta' (β '), sigma (s), and omega (w). However, the function of omega is unknown and is thought to possibly stabilize RNA polymerase.

In bacterial DNA, the promoter sequence is recognized by the sigma unit of the RNA polymerase. Upon recognition of the promoter sequence, the sigma factor will guide the RNA polymerase to the promoter. This sigma factor will then bind the RNA polymerase to the promoter through the α unit of the core polymerase.

Archaea
Archaeal RNA polymerases are pretty similar to eukaryotic RNA. Especially similar to RNA Polymerase II. These polymerases may have evolved from stripping down eukaryotic systems. An archea polymerase is used in PCR because it can withstand the high temperature used to split DNA strands.

Structure
RNA polymerase have a multitude of structural features that help in the transcription process. A Structure known as the clamp keeps the polymerase anchored to DNA. The flap ensures that the MRNA is retained. The rudder prevents DNA/RNA hybrid from occurring. DNA does not enter the mouth of the polymerase directly. It is usually held sidewise with a sharp bend to its left as it exits the polymerase. mRNA is believed to leave from the back of the polymerase. NTPs enter the active site as the same channel that DNA is pulled through but through a secondary channel.

Similarities and Differences between RNA Polymerase and DNA Polymerase
The synthesis of RNA and DNA is similar in many aspects. Both of them follow the synthesis direction of 5'->3'. Another is that the method of elongation is by the 3'OH group at the terminus of the growing chain that makes a nucleophilic attack on the innermost phosphate of the incoming nucleoside triphosphate. Another similarity is that the synthesis is driven by the hydrolysis of pyrophosphate. However the difference between the two is that RNA polymerase does not require a primer unlike DNA polymerase which does. Also although DNA polymerase can actually correct mistakes in the nucleotide transcription, RNA polymerase lacks this ability to excise the mismatches nucleotides.