Functional Analysis of the Three TATA Binding Protein Homologs in Methanosarcina acetivorans

Abstract

The roles of three TATA binding protein (TBP) homologs (TBP1, TBP2, and TBP3) in the archaeon Methanosarcina acetivorans were investigated by using genetic and molecular approaches. Although tbp2 and tbp3 deletion mutants were readily obtained, a tbp1 mutant was not obtained, and the growth of a conditional tbp1 expression strain was tetracycline dependent, indicating that TBP1 is essential. Transcripts of tbp1 were 20-fold more abundant than transcripts of tbp2 and 100-to 200-fold more abundant than transcripts of tbp3, suggesting that TBP1 is the primary TBP utilized during growth. Accordingly, tbp1 is strictly conserved in the genomes of Methanosarcina species. ⌬tbp3 and ⌬tbp2 strains exhibited an extended lag phase compared with the wild type, although the lag phase for the ⌬tbp2 strain was less pronounced when this strain was transitioning from growth on methylotrophic substrates to growth on acetate. Acetate-adapted ⌬tbp3 cells exhibited growth rates, final growth yields, and lag times that were significantly reduced compared with those of the wild type when the organisms were cultured with growth-limiting concentrations of acetate, and the acetate-adapted ⌬tbp2 strain exhibited a final growth yield that was reduced compared with that of the wild type when the organisms were cultured with growth-limiting acetate concentrations. DNA microarray analyses identified 92 and 77 genes with altered transcription in the ⌬tbp2 and ⌬tbp3 strains, respectively, which is consistent with a role for TBP2 and TBP3 in optimizing gene expression. Together, the results suggest that TBP2 and TBP3 are required for efficient growth under conditions similar to the conditions in the native environment of M. acetivorans.The basal transcription machinery of the Archaea resembles that of the RNA polymerase II system in the Eukarya domain that includes two essential general transcription factors, TATA binding protein (TBP) and transcription factor B (TFB) (3-5, 16, 25, 42). In order to establish promoter-directed transcription, TBP binds a TATA box located ϳ25 bp upstream of the transcription start site. The binding of TBP to the promoter allows TFB to bind at sites both upstream and downstream of the TATA box. TFB then recruits RNA polymerase to the promoter to establish the preinitiation complex, which is followed by transcription (2).Genes encoding multiple homologs of TBP and/or TFB have been identified in the genomes of several species in the domain Archaea. It has been proposed that the function of the homologs is to direct gene-specific transcription, analogous to the function of alternative factors specific to the domain Bacteria (1). Experimental evidence that supports this hypothesis has been reported for the archaeon Halobacterium sp. NRC-1, which contains six tbp genes and seven tfb genes (9, 12). A comprehensive systems approach provided evidence suggesting that there is global gene regulation by specific TFB-TBP pairs (12). In another study, two different mutants in which there was deletion of either the tbpD or tfbA h...

Genome-wide gene expression and RNA half-life measurements allow predictions of regulation and metabolic behavior in Methanosarcina acetivorans

“…Our data is consistent with a previous study showing that the primary TATA binding protein (TBP; tbp1 ) transcript was similarly expressed under the three growth conditions [75]; however, it differs for comparisons of accessory TBPs wherein our data suggest that tbp2 and tbp3 are expressed at similar levels to each other, while the previous report showed that the latter of the two was much less highly expressed. Both [75] and our study show that tbp3 is more highly expressed during methylotrophic than acetotrophic growth (almost identical fold changes in both studies), and this is supported by the observation that genes in amino acid metabolism and methylamine metabolism are differentially expressed upon its knockout as seen previously.…”

“…Both [75] and our study show that tbp3 is more highly expressed during methylotrophic than acetotrophic growth (almost identical fold changes in both studies), and this is supported by the observation that genes in amino acid metabolism and methylamine metabolism are differentially expressed upon its knockout as seen previously. Discussion of four additional transcription factors can be found in Additional file 1: Section “ Differentially expressed genes ”.…”

Same same but different: The evolution of TBP in archaea and their eukaryotic offspring

“…Seven specific pairs of TFB and TBP (out of the 42 possible combinations) were identified by co-immunoprecipitation and ChIP-seq experiments 24,30 . In a few archaeal species such as Methanosarcina , multiple TBP paralogues exist alongside a single TFB paralogue 27 . Without much variation in the TATA-box being possible and no specific TBP–TFB pairs, how might the multiple TBPs in Methanosarcina achieve their role in transcription regulation?…”

Transcription Regulation in Archaea

“…In vivo studies have shown that Archaea must retain at least one gene encoding TBP and one gene encoding TFB, although many archaeal species encode multiple TBP and TFB isoforms (6,21,(30)(31)(32)(33)(34)(35). Some differences in promoter sequence preferences and protein pairing have been noted in TBP-TFB isoform pairs (36)(37)(38)(39)(40)(41), but these minor differences are not on par with the clear but not always radical promoter sequence differences noted for alternative factors in bacterial transcription (39,42).…”