The formation of RNA is vital in the expression of genetic information. Transcription is the process in which DNA is converted into RNA so that it can be further processed into proteins. The enzymes RNA polymerase I, II, and III are found to have a major role in transcription of eukaryotes, which this discussion will focus on the second RNA polymerase which is responsible for the transcription of DNA into mRNA used to code proteins6.
Polymerase I transcription occurring in the nucleolus results in a precursor that codes for three RNA factors of the ribosome. The three rRNAs produced are 18S, 28S, and 5.8S, which are all components of the small and large subunit. 18S rRNA is involved with the small subunit (40S) and the 28S and 5.8S rRNAs are involved with the large subunit (60S). RNA polymerase 1 is thought to bind to the DNA with the help of a TATA-like sequence named the ribosomal initiator element. Along with this initiator element, an upstream promoter element located 150-200 bp upstream of the start site helps recruit the polymerase 1 to transcribe the ribosomal DNA. This pre-rRNA then undergoes splicing and further processing before it is released
Polymerase III is found in the nucleoplasm and transcribes tRNA. These tRNAs have the 5’ leader cleaved along with the removal of the 3’ trailer, finally CCA is then added by a CCA-adding enzyme. Polymerase III binds to DNA with the help of promoters that are downstream of the start site within the specific transcribed gene. The pre-tRNAs transcribed with the help of RNA polymerase III undergo further modifications and processing to ensure proper function.
Polymerase II has been studied with great detail recently with the help of Kornberg and his colleagues. By obtaining detailed crystallized structures of this enzyme, researchers are able to perform more extensive studies on mechanism based on the structural details. This website will focus primarily on structure, function, and mechanism of RNA polymerase II, which is responsible for the transcription of all protein-coding genes.
Initiation begins with the assembly of the main processing units, the RNA polymerase. The polymerase II enzyme is the central core of the transcriptional machinery in eukaryotic cells consisting of 12 subunits. The largest subunit, Rpb1 contains the active site of the enzyme that is combined in a single fold of the polymerase with the second largest subunit, Rpb2. Rpb2 forms the hybrid-binding region, and both Rpb1 and Rpb2 form the active center of the enzyme, which constitutes almost half of the total mass (>0.5 MD)6. Rpb3, Rpb10, Rpb11, and the Rpb12 subunits are all involved in polymerase II assembly. Rpb1, the “lobe” of Rpb2, and Rpb9 form the “upper jaw” region contained in the “jaw-lobe” component. The “shelf” module harbors the “lower jaw” which consists of Rpb5, Rpb6, and the “foot” and “cleft” regions of Rpb16. The Rpb4 and Rpb7 subunits are thought to be involved in a complex that recruits a dephosphorylation molecule, Fcp1, which dephosphorylates the carboxyl terminal domain of the polymerase II complex to end transcription allowing the RNAP to rejoin another initiation complex1. The specific subunit names button will display the region in the eukaryotic polymerase.
Transcription factors then bind to these promoter elements starting with the binding of transcription factor II D (TFIID) to the TATA box. This TFIID complex is 700-kd which includes the 30-kd TATA-box-binding protein. The surface of this binding protein contains docking sites for the binding of the other transcriptional components. TFIIA binds first, followed by TFIIB, TFIIF, RNA polymerase II, TFIIE, and lastly TFIIH form the basal transcription apparatus. During initiation, the carboxyl terminal domain (CTD) of polymerase II is unphosphorylated and interacts with an enhancer associated complex called mediator. There are many subunits of the mediator complex that are found to be important factors in the regulation of transcription5. Phosphorylation of the CTD occurs with the binding of the TFIIH factor, which marks a transition from initiation to the elongation stage. This phosphorylation stabilizes transcription elongation and will recruit RNA-processing enzyme that are vital for elongation.
Polymerase II binds to promoters that help coordinate binding to the correct initiation site on the DNA. Three main elements are found to be involved in the promoter region of RNA polymerase II. The TATA box is the most commonly recognized factor and is located around 30-100 bp upstream of the start site. A mutation of a single base in this TATA region impairs the promoter activity, proving that a specific sequence of AT pairs is necessary. An initiator element is often associated with the TATA box, which is located around -3 to +5 bp around the start site. This is the sequence that defines the starting site, and has been found to increase transcriptional activity. The third element is a downstream core promoter element that is commonly found to associate with the initiator element when the TATA box is not present. This promoter is found downstream of the start site between positions +28-+32.
During the initiation of transcription, Rpb1 and Rpb2 subunits form a cleft that harbors the DNA strand. This complex is anchored at one end by assembly of subunits; Rpb3, Rpb10, Rpb11, and Rpb12. The active site of polymerase II contains a magnesium ion bound on subunit Rbp1, which is referred to as metal “A”6. In single-subunit structures, metal A coordinates the 3-OH group at the growing end of the RNA and the a-phosphate of the substrate nucleoside triphosphate. Metal B (magnesium ion) coordinates the three phosphate groups of the triphosphate. Both metals stabilize the transition state during phosphodiester bond formation6. In Pol II, only metal A is persistently bound, at the upper edge of pore 1, whereas metal B is located further down in the pore, which may enter with the substrate and/or leave with the pyrophosphate after nucleotide addition. This pore is located in the floor of the cleft beneath the active site, allowing entry of substrate nucleotide triphosphates and also exit of RNA during retrograde movement of polymerase5.
Duplex DNA downstream of the active site is termed B-form DNA. This B-DNA lies in the Rpb1-Rpb2 cleft and follows a straight path to the active site. Rpb5, and regions of Rpb1 and Rpb9 on the opposite side of the cleft form “jaws” that grip the DNA and appear to be mobile and can open and close on the DNA7. The DNA will pass the active site containing the magnesium ion
During formation of the transcription complex there is a mobile element that is formed given the term “clamp”7. This clamp interacts with DNA from the active site of the DNA to about 15 residues downstream which will hold the template and transcript5. This clamp undergoes 5 different conformational changes in the “switch” regions, which are all found to interact with each other. Initially, the switch regions of this clamp are not very ordered, but will become ordered in the transcribing complex, suggesting that ordering is involved with the switch interaction with DNA downstream or in the DNA/RNA hybrid (see below).
As stated before, pore 1 is located below the active site of the enzyme, which contains both metal A and B. Pore 1 contains two nucleoside triphosphate binding sites, the entry (E) and nucleotide addition (A)14. It seems that NTP’s bind at the E site and will rotate around metal B to sample the base pairing in the A site. The positions of metals A and B vary slightly from one another, which proves the flexibility of the complex. Since the NTP is required to stabilize metal B, if the position of NTP changes, so will metal B. A protein-RNA interaction is vital at the beginning of elongation for phosphodiester bond formation. NTP must be held in the -1 and +1 region to form this phosphodiester bond, and will translocate to positions -1 and -2. The energy from base pairing alone isn’t sufficient to hold the dinucleotide product in the complex. Some Di and trinucleotides may be released from the complex, which will result in the abortive cycling of two to three residue fragments14. The flexability of the polymerase II complex provides insight that the active site is able to act under varying modes; as a polymerase, exonuclease, endonuclease, exopyrophosphorylase, and endopyrophosphorylase.
The eukaryotic polymerase is known to switch between forward (polymerization) and backward (backtracking) movement during transcription. This is a useful proofreading tool that can check for DNA damage, bound proteins, or natural pause sites in the DNA5. The transcriptional bubble holding the DNA-RNA hybrid will unzip the 3’ of RNA and will exit through pore 1, with the 5’ RNA single strand moving into the bubble. After too much backtracking the complex will be unable to restore the 3’ RNA to the active site which may result in the interaction with a funnel that is located in the pore where the 3’ RNA exits, and various elongation factors interact, such as TFIIS5. This factor contains a small zinc binding domain and a b hairpin with two conserved residues that has been found to be vital in RNA cleavage due to the close proximity of the conserved residues in relation to the active site of the polymerase.
The DNA/RNA hybrid is located in a region where the polymerase holds an unwound DNA strand containing 8-9 bp (transcription bubble)5. The base in the template strand at position +1 forms this hybrid. This is found between the bridge helix and an element of Rpb2 that blocks a linear DNA-RNA hybrid termed the “wall”. Due to this wall and the active site location, the hybrid must be tilted relative to the downstream DNA axis. The upstream end (5’) of the DNA-RNA hybrid must separate strands. RNA strands are seen to enter a binding site on the protein, which is located around 15 nucleotides upstream of the active site. Two grooves in the polymerase II structure which are shown to have function with the exiting of the hybrid6. RNA exits through the groove in the base, and the binding of RNA in this groove prevents the clamp from releasing the DNA, providing a lock on the closed clamp structure.
There is a point when newly synthesized RNA will separate from the DNA/RNA hybrid and exit from the polymerase molecule to be translated and the double stranded DNA can reform7. The two strands begin to separate at position -9 upstream of the currently open nucleotide. There are three protein loops from the RNA polymerase that appear to play an important role in the separation process. These loops are located in subunits Rpb1 (the lid, and rudder), and Rpb2 (the fork loop)7. The lid has the first important role as it wedges between the two strands beginning at the -9 position and then maintains the separation at position -10. The rudder is involved in maintaining the stability of the single stranded DNA at positions -9 and -10.
This occurs in a groove that has been localized near the clamp mechanism. The RNA will exit through this groove and then will be processed through splicing of introns and capping mechanisms5. First the 5’ end of the newly synthesized RNA chain is instantly modified. This will form a 5’-5’ triphosphate linkage with a GTP molecule, forming a cap. These caps are found to contribute to stability of the RNA by protecting the 5’ ends from certain phosphates and nucleases. The 3’ of the “pre”-mRNA is modified with the addition of a poly(A) tail8. This is added after transcription has ended and can span up to 250 adenylate residues. The specific function of the poly(A) tail is unknown, but there is evidence to suggest that it is involved in the enhancement of translation efficiency and the stability of mRNA.
Termination is defined as the permanent discontinuation of RNA synthesis followed by polymerase-DNA dissociation8. It has been demonstrated that termination elements located in the 3’ region of genes coordinate temporal and spatial interactions between the poly(A) site, nascent transcript, and the DNA template. Thus, the strength of the promoter, poly(A) site, and the termination element of a gene must interact to bring about the most efficient termination.