The narrative below is followed by references. Please note that these are limited to those papers that have been contributed by this research group only. References to the works of others can be found within these cited publications. Also note that although the figures depicted on this page represent the cumulative work of many people, they were modeled to focus attention on areas of most interest to this research program to assist us in the conceptualization of new experiments.

Any questions, comments or discussion regarding the models below or any aspect of this page would be welcomed by Rich Maraia at maraiar@mail.nih.gov

I have found that Internet Explorer resolves the figures on this page better than Netscape.

Mechanisms involved in eukaryotic transcription and other aspects of RNA expression are studied in the Section on Molecular and Cell Biology, in which RNA polymerase III (pol III) serves as a model system of eukaryotic transcription. Pol III produces small RNAs, including 5S ribosomal, transfer, and U6 small nuclear RNAs that are essential for cell growth.

During the past twenty years workers in several laboratories have uncovered the mechanism by which pol III is directed by ancillary transcription initiation factors to its target genes. These transcription factors (TF) recognize the promoters of the target genes and mediate assembly of the pre-initiation complex. For tRNA, B1-Alu and VA RNA genes these are the TFs IIIB and IIIC. These genes contain internal promoters, i.e., they reside within the transcribed region, that consist of A box and B box elements. TFIIIC recognizes the B box in a sequence-specific manner, making several contacts along the length of the gene. The A box-associated components of TFIIIC then recruit TFIIIB to bind to the DNA just upstream of the transcription start site. TFIIIB, and to lesser degree, TFIIIC then recruit the pol III enzyme in an orientation-specific manner to the start site of transcritpion. TFIIIA is a gene-specific factor, recognizing the internal control region (ICR) of the 5S rRNA gene. Other genes use different promoter types and different sets of TFs to recruit pol III (see Figure). Once formed, pol III transcription complexes are very stable and can recruit the pol III enzyme repeatedly for multiple rounds of transcription. With few exceptions, the subunits of TFIIIB, TFIIIC and pol III itself show evolutionary conservation from yeast to human, as has the promoter archectiture of tRNA and 5S rRNA genes. The mechanistic details of the assembly and structure of the pol III initiation transcription complex, as well as the identification and sequences of the TFs themselves, has been worked out, in large part, by workers in several laboratories, including those of E. Peter Geiduschek (UCSD), Robert G. Roeder (Rockefeller University), Arnold J. Berk (UCLA), and Andre Sentenac (CEA). The pol III complexes depicted below reflect their efforts; we are grateful to be able to use them here, and do so for illustrative purposes.

Transcription termination is a central focus of our research as it is a point at which several projects intersect. Termination by pol III begins with recognition of a specific termination signal in the DNA template, a stretch of several consecutive dA residues (dTs in the non-template strand), and ends with release of the nascent transcript product and disengagement of the polymerase. For pol III, efficient termination contributes to the overall high efficiency with which pre-assembled transcription complexes can be recycled for multiple rounds of RNA synthesis. Pol III itself contains ~17 polypeptide subunits, and has the intrinsic ability to recognize oligo(dA) as a termination signal. Evidence from the human-derived pol III system suggests that the efficiency of termination is increased by TFIIIC and other pol III-associated factors. La is a general factor that associates with all of the transcripts synthesized by pol III. This is achieved by high affinity binding to the common UUUOH motif at the 3' ends of these transcripts (see Figure). This motif represents a RNA copy of the termination signal and is a result of pol III termination. Evidence from three research groups indicate that the human La protein increases transcription termination efficiency. In a simple model, La would facilitate prompt release of the terminated transcript and in this way prepare the template for another cycle of transcription. However, in addition to this activity, it appears that the human La protein also plays a distinct and more active role in reinitiation. In any case, La remains associated with nascent pol III transcripts after their release and protects them from nucleolytic degradation. La then dissociates from the transcript and the latter associates with other proteins that are specific for that particular RNA (e.g., 5S rRNA associates with the ribosomal protein, L5, while U6 snRNA associates with the Sm components of the splicesome). Thus, La serves as a molecular link between transcription termination and the posttranscriptional handling or maturation of the pol III transcript. For some pol III products, the posttranscriptional processing phase of expression can be extensive (see Expression of an eukaryotic tRNA below).

Once assembled, pol III transcription complexes are stable and can undergo multiple rounds of transcription in a highly productive process known as recycling. We reported that the human La protein facilitates recycling (Maraia et al., 1994). Since La remains associated with the newly released transcript, free La molecules must then participate in recycling.

We mapped the phosphorylation site on human La to serine 366 and found that this modification inhibits La's ability to stimulate transcription recycling while dephosphorylation activates it (Fan et al., 1997). We also found another biochemical activity for La, recognition of the pppG- 5'-end of a nascent pol III transcript. Direct binding as well as nuclease protection assays using a variety of mutant La proteins in combination with a variety of mutant transcripts revealed that a short basic motif in the C-terminal domain of human La interacts with the 5'-pppG motif of the nascent transcript. The same region of La is required for transcriptional stimulation (Goodier et al., 1997). Moreover, phosphorylation adjacent to the short basic motif, on serine 366, also interferes with this activity. Therefore, both activities, 5'-pppG recognition and transcriptional stimulation are inhibited by serine 366 phosphorylation (Fan et al., 1997, Fan et al., 1998). One way to explain these results is that 5'-pppG recognition is used by La to stimulate transcription. The ability to recognize the initiating end of a nascent RNA would contribute to La's activity as a transcripton initiation factor (Maraia R., 1996). This is consistent with a finding from Robert Roeder's Laboratory. Those workers have demonstrated that La is a specific component of a human pol III holoenzyme (Wang et al, 1997).

We must now ask what happens upon termination by pol III and release of the RNA from the transcription complex? Our data indicate that unphosphorylated La does not readily dissociate from the nascent transcript. Phosphorylated La however, is disengaged from the 5'-pppG-end of the RNA, and in the case of precursor tRNAs, phosphorylated La facilitates 5'-end processing by RNase P (Fan et al., 1998). This model explains how one binding event, recognition of the 5'-pppG motif of a nascent pol III transcript, leads to two activities of La, stimulation of transcriptional initiation and posttranscriptional control of 5'-processing. Since only unphosphorylated La stimulates transcription, dephosphorylation would have to occur before La could be active for another round of RNA production. However, other possibilities exist. For example, the 3'-end of the RNA may be endonucleolytically processed while attached to La; this would separate La from the 3'-end of the transcript. Since it would be important for La to dissociate from the nascent transcript so that the latter can mature and function, there may be more than one way to ensure that La does disengage the nascent transcript.

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