Structural Biochemistry/RNA Polymerase III

RNA Polymerase III (also known as Pol III) is an enzyme that transcribes DNA to synthesize tRNA, 5S rRNA, and other small structural and catalytic RNAs. The transcribe RNAs are typically shorter than 400 base pairs. Like other polymerase reactions, transcription by Pol III involves three main stages: initiation, elongation, and termination. RNA Polymerase III has been most thoroughly studied in yeasts, but human Pol III have been identified and had its structure and function examined as well.

Initiation
Initiation by RNA Polymerase III can be divided into three classes depending on which genes correspond to specific transcription factors of Pol III.

Class I
Class I typically refers to transcription of 5S rRNA genes and requires TFIIIA. Pol III uses type I promoters, which consist of series of essential and non-essential regions for transcription. Typically, it requires an internal binding site composed of an A box, an intermediate, and a C box within the gene.
 * 1) TFIIIA binds to the internal control sequences, box A, intermediate, and box C.
 * 2) TFIIIA brings TFIIIC to the start site of the transcription, and TFIIIC binds to the DNA.
 * 3) Once TFIIIC is bound to the DNA, it brings TFIIIB to the upstream of the start site of transcription.

Class II
Class II refers to transcription of tRNA genes. It requires TFIIIB and TFIIIC. Pol III uses type II promoters which consist of two sequence blocks, box A and box B.
 * 1) TFIIIC binds to the internal control sequences, box A and B.
 * 2) TFIIIC brings TFIIIB to the upstream of the start site of transcription.
 * 3) TFIIIB assembles Pol III at the start site.

Class III
Class III involves transcription of U6 snRNA genes. Unlike class I and II, the type III promoter used by Pol III in this case is external to the gene. A TATA box is also present in type III promoter. Typically, TATA box is a distinct characteristic of type III promoters, but they are sometimes found in class II type II promoters of yeasts as well.
 * 1) SNAPc (SNRNA Activating Protein complex) binds to the PSE (Proximal Sequence Element), located upstream of the start site of transcription.
 * 2) SNAPc brings TFIIIB to the TATA box located upstream of the start site of transcription.

Termination
Pol III requires a simple sequence for termination: a run of T residues.

TFIIIA
TFIIIA (Transcription Factor for Polymerase III A) is a transcription initiation factor (TF) for transcription of 5s rRNA genes.

TFIIIB
TFIIIB (Transcription Factor for Polymerase III B) assembles RNA Polymerase III at the gene's promoter. It is positioned at the upstream of the start site of transcription. It consists of three subunits, TBP, Brf1, and Bdp1. It is stably bound to DNA, contributing to fast transcription rate of tRNA by re-initiation.
 * TATA-binding Protein (TBP): a protein that binds to TATA box of the DNA. It interacts with several other subunits of RNA Polymerase III, such as Tfc8 (TFIIIC). In humans, it interacts with TFIIIC, an analogue of TFIIIC found in yeasts.
 * TRF: a TBP homologue present in Metazoans (animals).
 * Brf1: a protein crucial for transcriptional activity of TFIIIB. In humans there are two homologs, HsBrf1 and HsBrf2.
 * Bdp1

TFIIIC
TFIIIC (Transcription Factor for Polymerase III C) is a TF for transcription of tRNAs. It consists of six subunits that are divided into two domains A and B, which attach to the A and B blocks (also called box A and box B) present in internal promoters.
 * Tfc1: binds to A-box of tRNA.
 * Tfc3: cooperates with Tfc6 to bind to the DNA.
 * Tfc4: a subunit of TFIIIC that interacts with Brf1 protein in TFIIIB to bring TFIIIB to the upstream of the transcription start site. It is part of domain A.
 * Tfc6: binds to the DNA.
 * Tfc7: binds to A-box of tRNA.
 * Tfc8: a subunit of TFIIIC serving as the flexible linker between the domains A and B which bind to the internal promoter boxes A and B.

Repressing Pol III Activity
RNA Polymerase III activity is largely affected by environmental changes, such as lack of nutrition, DNA damage, and other stress put on the cell. The main regulator for Pol III Activity is Maf1, a phosphoprotein.

Maf1 in Yeasts
Maf1 is the sole negative regulator for synthesis of tRNAs by Pol III in yeasts (also present in humans), such as Saccharomyces cerevisiae (Sc). In favorable growth conditions, tRNA molecules are produced at a rate of 3-6 millions of molecules in a single cell at 2-4 transcripts per gene, made possible by several rounds of re-initiation of Pol III by the initiation factor TFIIIB, which binds to the DNA. When growth conditions are favorable, Maf1 is phosphorylated, rendered inactive as binding between Maf1 and PolIII is inhibited, as Maf1 is exported from the nucleus, and as cytoplasmic Maf1 is inhibited from entering the nucleus. Phosphorylation is controlled by central cellular kinases casein kinase 2 (CK2) and TOR complex 1 (TORC1). CK2 is a protein kinase that encourages tRNA synthesis also by phosphorylating the TBP subunit of TFIIIB factor. Next, Maf1 is brought out of the nucleus of the cell, catalyzed by several factors. Protein kinase A (PKA) turns off nuclear localization sequences to prevent Maf1 from localizing around the nucleus. Then, a further interaction between Maf1 and Msn5 exportin finally transports Maf1 out of the nucleus into the cytoplasm. However, studies have shown that Maf1 is never fully excluded from the nucleus. Despite such thriving production, as environmental condition turns unfavorable for growth, such as when the cell is under stress or in starvation, the rate of tRNA synthesis drastically drops, repressed by Maf1.

When the condition is unfavorable, CK2, which usually phosphorylates Maf1, becomes inactive. The PP2A phosphatase dephosphorylates Maf1. Maf1 binds to the N-terminal of the largest subunit, C160, of RNA Pol III. Once Maf1 binds to C160, it changes the arrangement of Pol III subunits C82, C34, C31. Since they are required factors in transcription initiation, especially C34, Pol III is unable to interact with promoters as well as before. C34 relocation affects Pol III's interaction with Brf1 subunit of TFIIIB, as it is seen that free Pol III is able to bind the DNA and initiation factor, but Pol III-Maf1 cannot. MMaf1 is able to inhibit and stop the re-initiation of Pol III through inhibiting interaction between C34 subunit of TFIIIC and Brf1 subunit of TFIIIB.