RNA polymerase I transcribes only the genes for ribosomal RNA, from a single type of promoter. The transcript includes the sequences of both large and small rRNAs, which are later released by cleavages and processing. There are many copies of the transcription unit, alternating with nontranscribed spacers, and organized in a cluster (Figure 28.20).
The promoter has been best characterized in human cells, in which it consists of a bipartite sequence in the region preceding the startpoint (Figure AZ1). The core promoter surrounds the startpoint, extending from -45 to +20, and is sufficient for transcription to initiate. However, its efficiency is very much increased by the upstream control element (UCE), which extends from -180 to -107. Both regions have an unusual composition for a promoter, being rich in GC base pairs; and they are about 85% identical.
RNA polymerase I requires two ancillary factors. UBF1 (upstream binding factor 1) is a single polypeptide that binds to a GC-rich element in the core promoter and UCE. Factor SL1 (selectivity factor 1) does not by itself have specificity for the promoter, but once UBF1 has bound, SL1 can bind cooperatively to extend the region of DNA that is covered. Once both factors are bound, RNA polymerase I can bind to the core promoter to initiate transcription. We assume that factors bound at the core promoter interact directly with RNA polymerase I, but we do not know how binding of the same factors at the UCE stimulates initiation in the core region.
SL1 consists of four proteins. One of them, called TBP (TATA-binding protein), is a factor that is required also for initiation by RNA polymerases II and III. TBP does not bind directly to GC-rich DNA, so DNA-binding is probably the responsibility of the other components of SL1. It is likely that TBP interacts with RNA polymerase I, possibly with a common subunit or a feature that has been conserved among polymerases.
The behavior of SL1 resembles a bacterial sigma factor. As an isolated protein complex, it does not bind specifically to the promoter, but in conjunction with other components, specific promoter regions are bound. It may have primary responsibility for ensuring that RNA polymerase I is properly localized at the startpoint. A comparable function is provided for RNA polymerases II and III by a factor that consists of TBP associated with other proteins. So a common feature in initiation by all three polymerases is a reliance on a "positioning" factor that consists of TBP associated with proteins that are specific for each type of promoter.
A specific factor is required for RNA polymerase I transcription termination. It binds to an 18 base sequence.
Recognition of promoters by RNA polymerase III illustrates strikingly the relative roles of transcription factors and the polymerase III 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 startpoint. The promoters for snRNA (small nuclear RNA) genes lie upstream of the startpoint 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.
Before the promoter of 5S RNA genes was identified in Xenopus laevis (frog), all attempts to identify promoter sequences assumed that they would lie upstream of the startpoint. But deletion analysis showed that the 5S RNA product continues to be synthesized when the entire sequence upstream of the gene is removed!
When the deletions continue into the gene, a product very similar in size to the usual 5S RNA continues to be synthesized so long as the deletion ends before base +55. The first part of the RNA product corresponds to plasmid DNA; the second part represents the segment remaining of the usual 5S RNA sequence. But when the deletion extends past +55, transcription does not occur. So the promoter lies downstream of position +55, but causes RNA polymerase III to initiate transcription a more or less fixed distance away.
When deletions extend into the gene from its distal end, transcription is unaffected so long as the first 80 bp remain intact. Once the deletion cuts into this region, transcription ceases. This places the downstream boundary position of the promoter at about position +80.
So the promoter for 5S RNA transcription lies between positions +55 and +80 within the gene. A fragment containing this region can sponsor initiation of any DNA in which it is placed, from a startpoint -55 bp farther upstream. (The wild-type startpoint is unique; in deletions that lack it, transcription initiates at the purine base nearest to the position 55 bp upstream of the promoter.)
The structures of three types of promoters for RNA polymerase III are summarized in (Figure AZ2). There are two types of internal promoter. Each contains a bipartite structure, in which two short sequence elements are separated by a variable sequence. Type 1 consists of a boxA sequence separated from a boxC sequence, and type 2 consists of a boxA sequence separated from a boxB sequence. The distance between box.A and boxB in a type 2 promoter can vary quite extensively, but the boxes usually cannot be brought too close together without abolishing function.
Three accessory factors are involved at internal promoters. TFIIIA is a member of the zinc finger proteins. TFIIIB consists of TBP and two other proteins. TFIIIC is a large protein complex (>500 kD), comparable in size to RNA polymerase III itself, and contains at least 5 subunits.
We do not fully understand all the interactions that occur at the pol III promoters, but the principle is clear. At type 2 promoters, TFIIIC recognizes boxB, but binds to a more extensive region including both boxes A and B. At type 1 promoters, TFIIIA binds to a sequence that includes boxC, and this is required to enable TFIIIC to bind. In both cases, the binding of TFIIIC in turn enables TFIIIB to bind to a sequence surrounding the startpoint.
A crucial feature in defining the roles of the factors is that, at this point, TFIIIA and TFIIIC can be removed from the promoter (by high salt concentration in vitro) without affecting the initiation reaction. TFIIIB remains bound in the vicinity of the startpoint and its presence is sufficient to allow RNA polymerase III to bind at the startpoint. 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. This sequence of events explains how the promoter boxes downstream can cause RNA polymerase III to bind at the startpoint, farther upstream.
So TFIIIB functions as a "positioning factor," responsible for localizing RNA polymerase III correctly. Like SL1 at the pol 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. Recall that TFIIIB includes the same protein, TBP, that is present in SL1; this could be the subunit of TFIIIB that interacts directly with RNA polymerase III.
Although the ability to transcribe these genes is conferred by the internal promoter, changes in the region immediately upstream of the startpoint can alter the efficiency of transcription.
The upstream region has a more important role in the third class of polymerase Ill promoters, where there are three upstream elements. These elements are also found in promoters for snRNA genes that are transcribed by RNA polymerase II. (Genes for some snRNAs are transcribed by RNA polymerase II, while others are transcribed by RNA polymerase III.) The upstream elements function in a similar manner in promoters for both polymerases II and III. The TATA element appears to confer specificity for the type of polymerase.
Initiation at an upstream promoter for RNA polymerase III can occur on a short region that immediately precedes the startpoint and contains only the TATA element. However, efficiency of transcription is much increased by the presence of the PSE and Oct elements. The factors that bind at these elements interact cooperatively. (The PSE element may be essential at promoters used by RNA polymerase II, whereas it is stimulatory in promoters used by RNA polymerase III; its name stands for Proximal Sequence Element.)
The crucial TATA element is recognized by a factor that includes the TBP, the subunit that actually recognizes the sequence in DNA. The TBP is associated with other proteins, some of which are specific for promoters of the pol III type, and this may explain why RNA polymerase III is specifically recruited to these promoters. The function of TBP and its associated proteins is to position RNA polymerase III correctly at the startpoint.
There is an underlying unity in the functions of the factors at RNA polymerase III promoters. The factors bind at the promoter before RNA polymerase itself can bind. They form a preinitiation complex that directs binding of the RNA polymerase III. The alternative modes of promoter recognition by RNA polymerase III emphasize the importance of the factors for establishing the startpoint for initiation. RNA polymerase Ill 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 startpoint. For the type I and type 2 internal promoters, the assembly factors ensure that TFIIIB (which includes TBP) is bound just upstream of the startpoint, 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 startpoint in order to direct binding of RNA polymerase III.
RNA polymerase III terminates transcription with U's immediately after a GC-rich region (but there is no requirement for a stem-loop structure, unlike the prokaryotic factor-independent terminator that has a GC-rich stem-loop). Termination usually occurs at the second U within a run of four U's, but some molecules terminate with 3 or even 4 U's.