Control of transcription arrest in intron 1 of the murine adenosine deaminase gene.

Abstract

Transcription arrest plays a key role in the regulation of the murine adenosine deaminase (ADA) gene, as well as a number of other cellular and viral genes. We have previously characterized the ADA intron 1 arrest site, located 145 nucleotides downstream of the transcription start site, with respect to sequence and elongation factor requirements. Here, we show that the optimal conditions for both intron 1 arrest and overall ADA transcription involve the addition of high concentrations of KCI soon after initiation. As we have further delineated the sequence requirements for intron 1 arrest, we have found that sequences downstream of the arrest site are unnecessary for arrest. Also, a 24-bp fragment containing sequences upstream of the arrest site is sufficient to generate arrest downstream of the adenovirus major late promoter only in the native orientation. Surprisingly, we found that deletion of sequences encompassing the ADA transcription start site substantially reduced intron 1 arrest, with no effect on overall levels of transcription. At the same time, deletion of sequences upstream of the TATA box had no significant effect on either process. We believe the start site mutations have disrupted either the assembly or the composition of the transcription complex such that intron 1 site read-through is now favored. This finding, coupled with the increase in overall transcription after highconcentration KCI treatment, allows us to further refine our model of ADA gene regulation.Control of transcription elongation plays an important role in regulating the expression of many eukaryotic and viral genes (for a recent review, see reference 57). Eukaryotic cellular genes which are regulated in this way include the protooncogenes c-myc (6, 7, 29, 36, 41, 58), c-fos (12, 21, 40), and c-myb (3, 55), as well as the genes encoding adenosine deaminase (ADA) (10,11, 23,26,37), epidermal growth factor receptor (18), and ribonucleotide reductase (8). A number of genes from Drosophila melanogaster as well, including the heat shock genes hsp70 and hsp26, show regulation at the level of transcription elongation (35,44,51). Viral transcription units also show this type of control, including those of human immunodeficiency virus (46, 54), adenovirus (42, 49, 56), polyomavirus (53), and the minute virus of mice (2,32,48). The diversity among these examples may indicate that control of transcription elongation is a widespread gene regulatory phenomenon.It has been shown in several cases that events occurring around the time of initiation govern the fate of the transcribing RNA polymerase complex. For example, in the Ul small nuclear RNA gene, 3' end formation is dependent on initiation from the U1 promoter (20,43). For the Drosophila hsp70 gene, it has been shown that elements in the promoter are capable of setting up paused transcription complexes, even on heterologous downstream DNA (35). In some cases, the presence or absence of certain cis sequences or trans-acting factors determines the fate of the elongating polymerase complex....

Post-transcriptional Regulation of Endothelial Nitric-oxide Synthase by an Overlapping Antisense mRNA Transcript

“…We do not favor this consideration given that we found no important differences between cell types in nuclear run-off signals or pol II distribution along genomic regions that spanned exon 2 to the terminal exon 26 of NOS3. These findings also provide evidence against cell-specific attenuation of transcriptional elongation, as described for adenosine deaminase (52,53), where pol II pauses or arrests in proximal portions of the gene in nonexpressing cells. Here nuclear run-off analysis revealed the existence of positive signals with 5Ј-but not 3Ј-probes.…”

Essential Contribution of Intron Sequences to Ca²⁺-Dependent Activation of <i>c-fos</i> Transcription in Pituitary Cells

“…Stimulation of transcription elongation at and beyond arrest or pausing sites has been shown for a number of genes, including human and mouse c-fos [13, 15, 16, 17], c-mos , c-myc , human L-myc [14], mouse TNF-α gene [18], and the human adenosine deaminase gene [19, 20]. Under nonstimulated conditions we show that mouse c-fos transcription in rat pituitary cells is initiated, but not elongated (fig.…”

“…Premature termination of the RNA polymerase II complex within coding sequences or introns have been reported for several eukaryotic cellular genes, including c-fos [13, 15, 16, 17], c-mos , c-myc , L-myc [14], TNF-α [18], adenosine deaminase [19, 20], smooth muscle α-actin [21]and transglutaminase type I [22]. …”

Tissue-specific Rescue Suggests That Placental Adenosine Deaminase Is Important for Fetal Development in Mice

“…A 2.8-kb genomic ADA HindIII fragment containing the endogenous polyadenylation sequences, intron 11, and ϳ2 kb of 3Ј-untranslated region and 3Ј flank was then inserted at the 3Ј end of the cDNA. Next, the endogenous ADA promoter was replaced with an ADA promoter containing a 36-bp deletion in the 5Ј-untranslated region (11). A 6.2-kb BamHI to EcoRI fragment from the plasmid ADACAT (12) was inserted at the 5Ј end of the construct.…”