A discrete region centered 22 base pairs upstream of the initiation site modulates transcription of Drosophila tRNAAsn genes.

Abstract

We have studied the mechanism by which 5'-flanking sequences modulate the in vitro transcription of eucaryotic tRNA genes. Using deletion and linker substitution mutagenesis, we have found that the 5'-flanking sequences responsible for the different in vitro transcription levels of three Drosophila tRNA5.' genes are contained within a discrete region centered 22 nucleotides upstream from the transcription initiation site. In conjunction with the A-box intragenic control region, this upstream transcription-modulatory region functions in the selection mechanism for the site of transcription initiation. Since the transcription-modulatory region directs the position of the start site and the actual sequence of the transcription-modulatory region determines the level of tRNAAsn gene transcription, the possibility is raised that the transcription-modulatory region directs a transcription initiation event similar to open complex formation at procaryotic promoters.In procaryotic organisms, initiation of RNA synthesis is controlled by small, conserved DNA sequences located 10 and 35 nucleotides upstream of the transcription initiation site. RNA polymerase holoenzyme recognizes and binds these regions in this first step of the pathway that leads to open-complex formation and transcriptional activation (5, 42). The three nuclear RNA polymerases of eucaryotes do not recognize promoters efficiently and, as a result, are unable to independently initiate gene-specific transcription (for a review, see reference 38). Instead, eucaryotic transcription is dependent upon promoter-specific transcription factors that recognize and bind discrete sequence elements. Current models suggest that the binding of these protein factors activates transcription by allowing each gene to be recognized by the appropriate polymerase. For RNA polymerases I and II, the known sites of transcription factor interaction are 5' of the site of transcription initiation (9,15,45). For RNA polymerase III-directed transcription of eucaryotic 5S RNA, tRNA and adenovirus VA RNA genes, the sites of transcription factor interaction are 3' of the transcription initiation site, within the sequences encoding the RNA (6,16,17,48,50). RNA polymerase III-directed transcription of 7SL RNA, 7SK RNA, and U6 RNA genes requires enhancerlike sequence elements for transcription factor interaction 5' of the transcription initiation site, similar to RNA polymerase II promoters (3,7,30,47).The intragenic control regions (ICRs) in tRNA genes, the A-and B-box ICRs, contain the DNA sequence determinants that direct transcription factor activities (reviewed in reference 40). For tRNA genes, the transcription factors bind to the gene to form a complex that is stable throughout multiple rounds of transcription initiation (4,23,36). This stable complex, comprised of tRNA gene and transcription factors, is considered to form the recognition substrate for RNA polymerase III.Transcriptional activity of tRNA genes is particularly sensitive to mutations in the A-and B-box ICRs. All of the po...

Adjacent pol II and pol III promoters: transcription of the yeast retrotransposon Ty3 and a target tRNA gene

“…After transfer, the membrane was washed for 15 min in transfer buffer, dried and baked at 80°C for 1.5 hr. The filter-bound RNAs were hybridized to the 32P-labeled sup2+b-specific oligonucleotide at 420C for [12][13][14][15][16] hr according to manufacturer's instructions. Quantitation of levels of sup2+b pre-tRNA from low molecular weight RNA samples was by analysis of slot blot hybridizations performed as described (19).…”

“…RNA polymerase II transcription of tRNA genes initiates in the 5'-flanking region and is directed by transcription factors, TFIIIB and TFIHIC, bound upstream and downstream of the initiation site, respectively (6)(7)(8). Although Ty3 insertions do not disrupt the internal promoter elements of the tRNA genes, per se, they do replace sequences in the 5'-flanking region, a region which has been shown to affect the expression of tRNA genes in vitro and in vivo (9)(10)(11)(12)(13). Due to the highly-regulated nature of the sigma promoter, the existence of Ty3 and sigma element insertions offered a unique opportunity to study the effects of altering the 5'-flanking sequences of the tRNA gene, as well as potential interactions of polymerase II and Im transcription units.…”

A TATA Element Is Required for tRNA Promoter Activity and Confers TATA-binding Protein Responsiveness in DrosophilaSchneider-2 Cells

“…Specifically, alteration of the natural sequences between 30 and 20 bp upstream of the transcription start site reduces the capacity of three different Drosophila tRNA genes (tRNA Arg (34), tRNA Val (18), tRNA Asn (16)) and a Drosophila 5 S RNA gene (35) to direct transcription in Drosophila extracts. Although these effects are pronounced, a common sequence motif responsible for the activity of the wild-type Ϫ30 to Ϫ20 regions is not apparent.…”

Saturation mutagenesis of theDrosophilatRNAArggene B-Box intragenic promoter element: requirements for transcription activation and stable complex formation

“…Transcription reactions contained 200 ng template DNA (wild type or mutant pArg) and 800 ng carrier pUC8, 15 (36). Reactions were incubated at 24°C and stopped after 90 min by the addition of 50 /1 of a solution containing 1 mg/ml proteinase K, 0.1% (w/v) sodium dodecyl sulfate in 30 mM HEPES-KOH, pH 8.0 (preincubated at 37°C, 20 min), and incubated at 37°C for 1 h. After phenol and chloroform extraction, transcription products were collected by ethanol precipitation, resuspended in a solution containing 80% (v/v) formamide, 0.05% (w/v) xylene cyanol FF, 0.05% (w/v) bromphenol blue, 1 mM EDTA, and electrophoretically resolved using an 8% (w/v) polyacrylamide gel (20:1 acrylamide:N,N'-methylenebisacrylamide) containing 8.3 M urea, 89 mM Trizma base, 89 mM boric acid, and 2 mM EDTA (36). Transcription was quantitated by liquid scintillation counting of excised radioactive bands dissolved overnight at 45°C in 0.2 ml of HC104 (60%v/v) and 0.4 ml of H202 (30% v/v).…”