The nuclei of all eukaryotic cells contain three different RNA Polymerases, designated I, II and III. Like the DNA Polymerase that catalyzes DNA replication, RNA Polymerases catalyze the formation of the phosphodiester bonds that link the nucleotides together to form a linear chain. The RNA Polymerase moves stepwise along the DNA, unwinding the DNA helix just ahead of the active site for polymerization to expose a new region of the template strand for complementary base-pairing. In this way, the growing RNA chain is extended by one nucleotide at a time in the 5’-to-3’ direction. The substrates are nucleoside triphosphates (ATP, CTP, UTP, and GTP); as for DNA replication, a hydrolysis of high-energy bonds provides the energy needed to drive the reaction forward. Each eukaryotic RNA Polymerase catalyzes transcription of genes encoding different classes of RNA. Transcription by RNA Polymerase-III produces small, sta¬ble RNAs including tRNAs, the 5S rRNA associated with the large ribosomal subunit, one of the snRNA (small nuclear RNAs) required for pre-mRNA splicing, and the 7S RNA associated with the signal recognition particle involved in secretion of proteins and the insertion of membrane-spanning proteins into cellular membranes. The functions of many other small RNAs produced by RNA Polymerase-III are as yet undiscovered.
Recognition of promoters by RNA Polymerase-III illustrates strikingly the relative roles of transcription factors and the polymerase enzyme. The promoters fall into two general classes that are recognized in different ways by different groups of factors. The promoters for 5S and tRNA genes are internal; they lie downstream of the start point. The promoters for snRNA genes lie upstream of the start point in the more conventional manner of other promoters. In both cases, the individual elements that are necessary for promoter function consist exclusively of sequences recognized by transcription factors, which in turn direct the binding of RNA Polymerase. The initial transcripts produced from tRNA genes are precursor molecules, which are processed into mature tRNAs; in this regard, tRNA genes are similar to pre-rRNA genes. The transcription-control regions of tRNA genes lie entirely within the transcribed sequence. Two such internal control regions are required. These DNA sequences encode the most highly conserved portions of eukaryotic tRNAs, the D-loop and the T*PCG loop. These two highly conserved sequences referred to as the A-Box and B-Box are for two distinct purposes: (1) to encode essential regions of tRNA molecules required for protein synthesis, and (2) to bind proteins necessary for transcription initiation by RNA Polymerase-III.
The DNA-binding factors required for RNA Polymerase-III to initiate transcription have been best characterized in Saccharomyces cerevisiae. Assembly of the Initiation Complex on tRNA genes begins by binding of TFIIIC to the A-Box and B-Box in the promoter (Ref.1). TFIIIB then binds =50 bp upstream of the A-Box. TFIIIB can bind to many different sequences at this position, indicating that it is directed to bind to DNA by its interaction with TFIIIC rather than by sequence-specific interactions with DNA. TFIIIC is composed of six polypeptides with a total molecular weight of -600 kDa. TFIIIB is made up of three subunits. One is TBP (TATA-Binding Protein), which is a subunit of a general initiation factor for all three nuclear RNA Polymerases (Ref.2). The second, called BRF (TFIIB-Related Factor) is similar in sequence to TFIIB, and performs a similar function in initiation by RNA Polymerase-III as TFIIB does for RNA Polymerase-II (Ref.3 & 4). The third subunit of TFIIIB is a 90-kDa polypeptide called B" (Ref.5). Once TFIIIB has bound, then RNA Polymerase-III can bind and initiate transcription in the presence of ribonucleoside triphosphates. Initiation by RNA Polymerase-III does not require hydrolysis of an ATP Beta-Gamma bond similar to RNA Polymerase-I. Once TFIIIB binds, TFIIIC can be removed without affecting initiation by RNA Polymerase-III. Thus, TFIIIC serves as an assembly factor for the critical initiation factor, TFIIIB.
In addition to TFIIIC and TFIIIB, transcription of 5S-rRNA genes requires a third initiation factor called TFIIIA. TFIIIA is a member of zinc finger proteins. Binding of this factor, which is a monomer, to the 5S-rRNA gene begins assembly of the Initiation Complex (Ref.6). The binding site for TFIIIA, called the C-Box is located 81-99bp downstream from the start site. It is an internal control region like the A and B-Boxes of tRNA genes. Synthesis of 5S rRNA is initiated by binding of TFIIIA to the C-Box. Once TFIIIA has bound, TFIIIC binds to the gene at a similar position relative to the start site. TFIIIB then binds, interacting analogously with TFIIIC as it does in a tRNA gene. Once TFIIIB has bound, RNA Polymerase-III binds and initiates transcription. TFIIIA thus acts as an assembly factor for binding of TFIIIC, which then performs the same function that it serves on tRNA genes as an assembly factor for TFIIIB.
RNA Polymerase-III does not itself seem to have a great intrinsic affinity for any particular sequence of DNA. It binds adjacent to factors that are themselves bound just upstream of the start point. For the internal promoters, the assembly factors ensure that TFIIIB (which includes TBP) is bound just upstream of the start point, to provide the positioning information. For the upstream promoters, transcription factors that directly recognize the upstream sites form a complex (including TBP) that is recognized by RNA Polymerase-III. So irrespective of the location of the promoter sequences, factor(s) are bound close to the start point in order to direct binding of RNA Polymerase-III. The presence of TFIIIB is sufficient to allow RNA Polymerase-III to bind at the start point. So TFIIIB is the only true initiation factor required by RNA Polymerase-III. TFIIIA and TFIIIC are assembly factors, whose role is to assist the binding of TFIIIB at the right location. So TFIIIB functions as a "positioning factor," responsible for localizing RNA Polymerase correctly. Like SL1 at the RNA Polymerase-I promoter, it resembles a Sigma Factor, in lacking the ability to bind DNA by itself, but being able to bind in conjunction with other proteins.