Molecular Genetics 

Facts

RNA polymerase I transcribes practically identical copies of a single gene, the rRNA precursor gene. Thus, the consensus sequence approach using different genes is not possible.  Cell free extracts have been tested for transcription from exogenously added rDNA templates with results in the table (ref).		rDNA template	extract source
			mouse	human
		mouse	++++	-
		human	-	++++
	Short segments of sequence in the region surrounding the start site of human rRNA transcription were replaced with other sequences by linker scanning mutagenesis. The activity of the mutated DNAs in stimulating transcription in a HeLa cell-free extract was measured (ref). The approach identified two regions (UCE and Core) as important for efficient transcription. Biochemical analysis revealed that UCE is a target for binding a protein factor UBF1 that binds the species-specific SL1 transcription factor (ref).

Interpretations

• Comparison of rDNA genes of different species does not generate a consensus promoter sequence since RNA polymerase I transcription initiation appears to be species specific.
• Species specificity also means that no generalizations about RNA polymerase I promoters are possible.
• Because rDNA transcription units are arranged in tandem, transcription of the upstream unit may influence initiation at the downstream unit.
• This is an example of the reverse genetics approach to the identification of genetic signals. In this approach the gene is changed in a defined way and then the effects of the change are determined.  In the genetics approach, mutant organisms with the change of interest are identified first.  Then, the mutations are characterized to determine how the genotype has changed.

Further information

• 5' flanking regions in mouse rDNA also are needed for proper initiation in this organism.
• The Drosophila rDNA promoter also is bipartite: residues -18 to +4 and -43 to -27 (ref).
• In yeast, a region 210 to 2230 bp upstream of the start site is needed for correct initiation (ref). A further upstream site produces a 15-fold enhancement, is orientation specific and position independent (ref).  
• The SL1 factor contains the TATA-binding protein (TBP) and three TBP associated factors (TAF's).  These four polypeptides are necessary and sufficient for SL1 activity (ref).
• DNase I footprinting has also been used to characterize the rRNA promoter region (ref).
• The reporter gene approach is a variation of the reverse genetics approach. Modified promoters are placed upstream of a reporter gene and their activities measured in transient expression assays.

This is page 2228 of Molecular Genetics by Ulrich Melcher, © 1997, 1998, 2002-3

E-mail inquiries to U. Melcher------------Last Updated: 11 September, 2003