Transcription of individual tRNA^Gly₁ genes from within a multigene family is regulated by transcription factor TFIIIB

Abstract

In eukaryotes, nuclear gene transcriptions are accomplished by three different RNA polymerases, RNA pol I, pol II and pol III [1,2]. The promoters for class III genes transcribed by RNA pol III, with the exception of the snRNAs, generally lack a TATA box but still require TATA box binding protein (TBP) for transcription [3][4][5]. The genes encoding tRNAs have promoter elements located within the coding region of the genes (designated as the A and B boxes), and require two basal factors, TFIIIB and TFIIIC [6], which are multisubunit proteins [7][8][9][10]. TFIIIC binds to the A and B boxes first, followed by recruitment of TFIIIB in the immediate upstream region (through protein-protein interaction) and finally the RNA pol III [11][12][13]. TFIIIB consists of three subunits, B-double prime 1 (Bdp1; 90 kDa), TFIIB-related factor 1 (Brf1; 60 kDa) and TBP in yeast, or two forms, TFIIIBa (comprising TBP, Brf2 and Bdp1 required for transcription of U6-type RNA pol III promoters) [14] and TFIIIBb (comprising TBP, Brf1 and Bdp1 required for transcription of tRNA and VA1-type RNA pol III promoters) [15], in humans. In the absence of TATA box sequences in these promoters, recruitment of TBP to the transcription site is achieved by interactions between the associated factors [16,17]. TFIIIB is analogous to the pol II-specific factor,

Different sequence signatures in the upstream regions of plant and animal tRNA genes shape distinct modes of regulation

“…TFIIIB is the essential factor that recognizes specific motifs in the upstream sequences of tRNA genes [preferably TA-rich regions in plants (28,43) and yeast cells (42)] and its binding stability to the cognate motif determines the expression level within one family of isodecoders (32). In human cells, expression of the genes encoding the three subunits of the TFIIIB complex, Brf1, Bdp1 and TBP, is either coordinated (45) or differentially modulated (46,47).…”

“…TFIIIB can directly be recruited to a typical TATA box even in the absence of TFIIIC and independently initiate transcription by pol III recruitment in yeast (30,31). In silkworms, TFIIIB binds stably to the promoter sequence, but is transcriptionally incompetent in the absence of TFIIIC (32). Importantly, TA-enriched regions located immediately upstream of the tRNA coding sequence (within the first 50 nt) enhance transcription, whereas multiple copies of such sequences in the far upstream regions reduce the tRNA transcription levels; sequestration of the TFIIIB complex at multiple binding sites decreases its effective concentration thus reducing the formation of the initiation complex with pol III (32).…”

“…In silkworms, TFIIIB binds stably to the promoter sequence, but is transcriptionally incompetent in the absence of TFIIIC (32). Importantly, TA-enriched regions located immediately upstream of the tRNA coding sequence (within the first 50 nt) enhance transcription, whereas multiple copies of such sequences in the far upstream regions reduce the tRNA transcription levels; sequestration of the TFIIIB complex at multiple binding sites decreases its effective concentration thus reducing the formation of the initiation complex with pol III (32). In addition, the regions upstream of the 5′-initial nucleotide of the mature tRNAs in plant and yeast (usually at positions −7 to −3) bear another conservative CAA motif which together with the TATA boxes cooperatively enhances transcription (28,33).…”

Transcriptional activation of a moderately expressed tRNA gene by a positioned nucleosome

“…In vitro transcription reactions were carried out in a final volume of 30 µl, 20 mM Hepes, pH 7.9, 60 mM KCl, 6 mM MgCl 2 , 0.1 mM EDTA, 6 mM creatine phosphate, 50 µM each of ATP, CTP and UTP, 10 µM GTP, 5 µCi of [α- 32 P]GTP (3000 Ci/mmol), PSG nuclear extract (4-8 µg of protein) and 100 ng of DNA template (naked DNA or chromatin), incubated at 30 • C for 1 h. Increasing amounts of purified TFIIIC fraction were added to the freshly assembled chromatin wherever indicated and were incubated for 20 min at 30 • C before the addition of the nuclear extract. For single-round transcriptions, incubations were carried out initially for 10 min in the absence of non-radioactive GTP and a further 50 min after adding 10 µM GTP and heparin (100 µg/ml) [30]. Competition for transcription factors was performed as in the standard transcription reactions at suboptimal concentrations of nuclear extract (4 µg of protein) so that the transcription factors were limiting and the transcription levels could be enhanced by external supplementation with the transcription factors TFIIIC or TFIIIB.…”

“…The stability of the interaction between TFIIIB and the tRNA Gly 1 -4 gene was examined by the formation of heparin-resistant complexes [30]. A 700 bp HindIII fragment from plasmid pBmH1 (containing full-length tRNA Gly 1 -4) or a 255 bp DraI fragment from the same plasmid (from − 145 nt to + 110 nt with respect to the tRNA Gly 1 -4 coding region) were radioactively labelled by end-labelling or random priming and were used as probes [29].…”