Detailed description of transcription
RNA polymerase II (Pol II) is the enzyme responsible for the conversion of DNA into mRNA in eukaryotes. Pol II is localized in the nucleus of cells where the first step of gene processing called transcription takes place. Pol II does not carry out transcription solely by itself a multitude of transcription factors (as many as six transcription factors) join forces with Pol II and assist in promoter recognition and localized melting of the DNA double helix6. Furthermore a mediator protein transfers information from activators and repressors to the transcription complex. The complete Pol II enzyme consists of 12 subunits and has a total mass of around 0.5 MD. Pol II contains over 3500 amino acids, 28,000 plus nonhydrogen atoms of which 8 are Zn2+ ions6. The core Pol II unit is comprised of 10 subunits. Rpb 1 and Rpb 2 are the two largest sububnits that comprise half the mass of the enzyme. 53,000 angstroms2 of surface area is buried in subunit interface and a third of that is found between Rpb1 and Rpb26. The active center of the enzyme is found in the regions of Rpb1 and Rpb2.four other subunits Rpb3, Rpb10, Rpb11, and Rpb12 anchor Rpb1 and Rpb2 and also assist in the assembly of Pol II. The “jaw-lobe” element is made-up of regions of Rpb1 and Rpb9 which houses the “upper jaw” and Rpb2 which contains the “lobe”6. Rpb5 houses the “lower jaw” and the “assembly” domain; Rpb6 and the “foot and “cleft” sections of Rpb1 make up the “shelf” element. The final subunit Rpb8 in the core Pol II comprises the “clamp” element. The “clamp” element is attached to Pol II through the cleft region of Rpb1 and the anchor region of Rpb2 and by a set of five switch regions in Rpb66. These switches allow for movement of the clamp. A loop nearest the active center is termed the rudder and is involved in the separation of DNA –RNA hybrids. A second and third loop make-up the lid and zipper respectively and assist the rudder in separation and maintenance. A “flap loop” which is made from a disordered loop on top of the wall also provides some maintenance capabilities. The complete Pol II has two smaller subunits Rpb4 and Rpb7 which aid the core Pol II in binding of the promoter and are not necessary for transcription 8. The surface charge of Pol II possesses mainly a negative charge except for a positively charged lining of the cleft, active center, the wall, and saddle (between the clamp and the wall).
To begin the process of transcription Pol II must bind to DNA. To do this the complete Pol II enzyme loosely binds to an unwound double helix of DNA and slides along the backbone in search of a promoter1. Eukaryotes posses many promoters upstream and downstream of the start site, when Pol II finds the correct promoter usually a TATA box it binds tightly to the DNA1. Rpb4 and Rpb7 dissociate from the complex leaving behind the core Pol II. DNA must now enter Pol II, small rotations of the jaw-lobe and shelf elements and a large swinging motion of the clamp create a straight opening for DNA through the cleft that permits entry of promoter DNA (which needs to remain straight)6. The DNA can now contact the jaw portion of Rpb9 and also fit into a concave surface of the Rpb2 lobe. Furthermore it passes over the saddle and comes into contact with switch 2, switch 3, the rudder, and the flap loop. The transcription factor TFIIB binds to the DNA on the backside of Pol II. It binds to a region corresponding to about 25 bps between the TATA box and the transcription start site and is thought to aid in start site determination6.Once the clamp has closed and DNA is bound to Pol II localized melting of about 17-20 bps of DNA by the adenosine-5’-triphosphate-dependent helicase activity of transcription factor TFIIH takes place6. The binding and melting of the DNA forms what is known as the transcription bubble1. The melted region of DNA passes near the active center and across the saddle. The single strand template DNA can bind to sites in the active center, on the floor of the cleft and along the wall. The switches close the clamp essentially locking DNA in Pol II, when the switches sense the presence of the DNA-RNA hybrid. The melting of DNA removes the double stranded DNA from the saddle to make room for the RNA which must cross the saddle to exit6.
Once binding and melting of the DNA have taken place the transcribing complex is now ready for elongation of the mRNA transcript. The DNA passes through as set of elements termed jaws and is held in place by the clamp. A hole in the floor of the active center allows for entry of nucleoside triphosphates and exit of RNA upon retrograde movement for corrections6. The positively charged region of the active center helps to move DNA into the active center without expending much energy. A Mg2+ ion found in the active center helps coordinate the 3’ –OH group of the growing end of the newly formed RNA and the α-phosphate of the incoming nucleoside triphosphate. A second Mg2+ ion helps coordinate the phosphates on the incoming nucleoside triphosphate6. Both of the metals together help to stabilize the formation of the phosphodiester bond. A structure known as the bridge helix which is straight in Pol II has been implicated in the process of translocation of the complex during longation. The bridge helix maintains contacts with the DNA-RNA hybrid during the elongation process and allows Pol II to translocate along the DNA without dissociation of the enzyme from the DNA6. As Pol II moves along the DNA and the subsequent DNA-RNA hybrid grows the RNA must somehow dissociate from the hybrid. The clamp with the assistance of the rudder takes care of this by sensing the hybrid in the upstream portion of the transcription bubble and seperates the strand. Once separated the newly formed RNA must exit the Pol II complex, it achieves its exit through the base of the clamp at an area known as “groove 1”6. RNA about 15-18 bps long lies on the saddle which is part of groove 1, the saddle is positively charged which allows it to interact with nucleic acids of RNA. Once the RNA strand is about 25 bps long the process of capping begins so as to avoid degradation by nucleases1. RNA’s exit from groove 1 is located near residue L1450 of Rpb1 which is located near the carboxy terminal domain (CTD). The CTD of Rpb1 is the site where RNA processing enzymes interact with the phosphorylated form of the CTD6.
The final step in eukaryotic transcription is the process of termination. This process is not well understood in eukaryotes. It is known that two things are essential to Pol II termination. One being a 3’ formation end signal close to the poly (A) site and the other a downstream element found near the termination signal7. The strength of the poly (A) site determines how effective termination will be. Moreover the downstream element is thought to make Pol II pause at this downstream element7. The weak interaction of the U-A base pair allows for dissociation of the RNA –DNA hybrid and the termination causes the Pol II to pause. This pause allows termination factors to catch the Pol II complex and then breakdown the Pol II complex7. The pre-mRNA is then further modified by the addition of the poly(A) tail and introns are excised forming a mature mRNA product with a cap and a tail1.