INTRODUCTION
Transcription in archaea: a mosaic of eukaryotic and bacterial features
1.1.1 Basal transcriptional machinery in archaea
Forty years ago, based on the 16s rRNA gene sequences, archaea were recognized as the third domain of life next to bacteria and eukaryotes (Woese et al., 1990; Woese & Fox,
Since the 1977 milestone in evolutionary biology, numerous studies have highlighted the distinct characteristics of archaea, establishing them as a separate domain Research has shown that archaea possess a transcription apparatus that closely resembles a simplified version of the eukaryotic RNA polymerase II system.
& Werner 2011; Orell et al 2013; Karr 2014; Gindner et al 2014; Kessler et al 2015)
Research on archaeal RNA polymerase (RNAP) from Sulfolobus acidocaldarius suggests that archaea may initiate transcription similarly to eukaryotes Archaeal RNAP is a highly conserved protein complex made up of 13 subunits, closely related to eukaryotic RNAP II The canonical core promoter in archaea features a TATA box, an AT-rich region located approximately -26 to -30 bp upstream of the transcription start site (TSS), along with a purine-rich transcription factor B recognition element (BRE) just upstream of the TATA box Additionally, archaeal promoters may contain less defined elements like the initiator element (INR) and promoter proximal element (PPE), although their presence varies among different archaeal groups For instance, while the INR is often undetectable in haloarchaeal promoters, it is prominent in methanogens and Sulfolobales The PPE, found primarily in Sulfolobus promoters, is located between the TATA box and the TSS.
Transcription factors are essential for the recruitment of RNA polymerase II (RNAPII) and RNA polymerase (RNAP) to promoters, as they cannot bind independently Notably, three types of transcription factors have been extensively studied in the context of archaeal transcription initiation: TATA-binding proteins (TBPs), transcription factor B (TFBs), and others.
Transcription factors E (TFEs) play a crucial role in the transcription process, with TBP and TFB being essential for promoter-specific transcription in vitro, as established by Bell et al in 1998 The assembly of TBP and TFB at the promoter site is vital for recruiting RNA polymerase (RNAP) to the transcription start site (TSS), leading to the formation of the pre-initiation complex (PIC) TFE enhances transcription initiation by improving TATA-box recognition, according to Bell et al in 2001, while further research by Grünberg et al in 2007 revealed that TFE also stabilizes the transcription bubble during elongation Notably, these three archaeal transcription factors are homologous to their eukaryotic counterparts, TBPs, TFBII, and TFEα.
& Jackson 2000; Thomas & Chiang 2006; Werner & Weinzierl 2005; Grove 2013)
Archaeal TBPs share a high degree of similarity with the C-terminal domain of eukaryotic TBPs, both in structure and function, with archaeal TBPs consisting of approximately 180 amino acids and featuring two direct repeats that are about 40% identical Transcription initiation occurs when TBP recognizes and binds to the TATA box, facilitating promoter bending and the recruitment of TFB to the BRE site Recent studies highlight differences in the TBP–DNA interaction lifetimes between archaeal and eukaryotic systems, revealing that eukaryotic interactions involve a linear, two-step bending mechanism, stabilized by TFBII, while archaeal promoter bending by TBP occurs in a single step and requires TFB Notably, the role of TFB in stabilizing the TBP–DNA complex may not be a universal mechanism but rather a specific function that enhances specificity among archaeal TATA-containing promoters.
TFBs in archaea exhibit notable similarities to eukaryotic TFIIB, featuring an N-terminal domain of 100-120 amino acids and a C-terminal domain with two repeat sequences of approximately 90 amino acids The C-terminal core domain and the helix-turn-helix (HTH) motif of TFB are crucial for its interaction with TBP and for binding to the BRE Recent studies, including the crystal structure analysis of TBP and the C-terminal core of TFB from P woesei, have revealed detailed stereo-specific interactions between the BRE and the HTH motif in TFBc, particularly in the context of a promoter containing a TATA-box and BRE.
The interaction between TFB and TBP-DNA is crucial for recruiting RNA polymerase (RNAP) to the transcription start site (TSS) The N-terminal domain of TFB contains a zinc ribbon motif that facilitates its interaction with RNAP Unlike in eukaryotes, the opening of archaeal promoters occurs without the need for energy.
& Thomm, 2001; Yan & Gralla, 1997) Archaeal TBP and TFB alone are capable of assisting RNAP in the formation of the transcription bubble (Hausner & Thomm, 2001)
The crystal structure of the archaeal ternary complex, comprising the TATA-binding protein (TBP), transcription factor B (TFBc), and a TATA-box and BRE-containing oligonucleotide, illustrates the molecular interactions essential for transcription initiation (Littlefield et al., 1999; PDB Acc No 1D3U) In archaea, the formation of a pre-initiation complex (PIC) begins with TBP binding to the TATA box, followed by TFB's interaction with the TBP-DNA complex, which then recruits RNA polymerase (RNAP) and transcription factor E (TFE) Additionally, transcriptional repressors inhibit transcription by binding to core promoter-overlapping sequences, blocking access for TBP, TFB, and RNAP during PIC assembly, while transcriptional activators enhance PIC formation by binding to sequences upstream of the BRE (Peeters et al., 2015).
Archaeal TFE is a homolog of the α subunit of TFIIE TFE was shown to associate with RNAP and stimulate DNA melting (Grünberg et al., 2007) The βTFE in archaea was
Introduction recently identified and shown to interact with TFE (αTFE) but βTFE are not conserved in all Archaea (Blombach et al., 2015)
1.1.2 Regulation of transcription in archaea
Transcription regulation is crucial at every stage, but controlling gene expression is most effectively achieved during the initiation phase This process involves key components such as promoter elements, general transcription factors, and gene-specific regulators (Balleza et al., 2009; Gehring et al., 2016; Karr, 2014).
1.1.2.1 Regulation of transcriptional initiation by general transcription factors
Transcription regulation by general transcription factors (GTFs) is crucial for gene expression control In bacteria, sigma (σ) factors play a key role in adapting gene expression to environmental stresses Eukaryotes utilize four different RNA polymerases (RNAPs) and various GTFs for global transcription regulation Most archaeal genomes contain at least one TATA-binding protein (TBP) and one transcription factor B (TFB), with some archaea like Halobacterium NRC-1 having multiple paralogs, which can theoretically create numerous TFB-TBP pairs for transcription initiation Research has shown that Halobacterium NRC-1 employs seven of these pairs, with TBPe being the most frequently interacting TBP Additionally, Methanosarcina acetivorans has three TBP homologs and one TFB, illustrating the diversity of transcription regulation mechanisms in archaea.
In the studied organism, TBP1 is more influential in gene expression compared to TBP2 and TBP3 Nonetheless, TBP2 and TBP3 are crucial for achieving optimal growth in conditions with limited acetate availability.
1.1.2.2 Regulatory motifs in archaeal promoters
Regulators play a crucial role in gene transcription by attaching to specific DNA sequences known as binding motifs within promoters The positioning of these binding sites relative to the core promoter, including the TATA box and BRE, influences whether the regulator acts as a repressor or an activator (Peeters et al., 2013) Typically, archaeal repressors bind to sites that overlap with or are located downstream of the BRE and TATA box, thereby inhibiting transcription.
The formation of the pre-initiation complex (PIC) is inhibited by factors that prevent the binding of TBP, TFB, or RNAP to promoters (Bell et al 1999; Dahlke & Thomm 2002; Lee et al 2008; Karr 2010; Keese et al 2010) In contrast, activators typically bind to sequences located upstream of the core promoter (Kessler et al., 2006; Ochs et al., 2012; Ouhammouch et al., 2003; Peng et al., 2009b) Despite this, the identification of activators in archaea and the understanding of their mechanisms remain limited compared to the extensive knowledge of archaeal repressors (Gehring et al., 2016; Karr, 2014; Peeters & Charlier, 2010).
Transcription regulators are crucial in gene expression, with their binding sites on promoter regions identified as regulatory motifs Most of these motifs act as binding sites for repressors, including the TGM (Thermococcales-glycolytic-motif) in Thermococcales, as well as MDR1, NrpR, and Phr Conversely, some motifs, such as those for ArnR, ss-Lrp, Ptr2, and MalR, function as binding sites for activators Notably, the ARA box, associated with the arabinose-binding protein (AraS), is recognized as an activating element, despite the absence of its regulator A common characteristic among these regulatory motifs is the semi-palindromic sequence, often featuring less or non-conserved base pairs at the center An exception to this pattern is the binding site of the heat shock regulator Phr in Pyrococcus furiosus, which is a non-palindromic sequence.
1.1.2.3 Gene-specific transcriptional regulators: repressors, activators
Archaeal genomes, despite having a eukaryotic-like basal transcription machinery, are characterized by a predominance of bacterial-like regulators rather than eukaryotic ones Many of these archaeal regulators resemble bacterial proteins with helix-turn-helix (HTH) domains, while others feature the ribbon-helix-helix (RHH) motif or the zinc ribbon Transcriptional regulators with HTH motifs typically function as dimers or higher oligomers, binding to palindromic DNA sequences and interacting with two adjacent major groove segments on the same side of the DNA helix.
The DNA damage response in hyper-thermophilic archaea
1.2.1 The DNA damage response (DDR)
Bacteria and eukaryotes have developed advanced DNA damage response (DDR) pathways to preserve genome integrity against DNA damage These DDR mechanisms swiftly identify DNA lesions, activate repair proteins, and temporarily halt DNA replication and cell division until the repair process is fully completed.
The SOS response is a critical DNA damage response (DDR) mechanism in bacteria, involving over 40 independent genes regulated by the LexA repressor and RecA inducer Under normal conditions, LexA binds to the SOS box in the promoter region of SOS genes, inhibiting their expression However, in the presence of significant DNA damage, RecA activates LexA self-cleavage, allowing the expression of SOS genes, which include those coding for DNA repair enzymes, division inhibitors, and the mutagenic DNA repair polymerase Pol V Additionally, the RecA filament facilitates the invasion of homologous double-stranded DNA sequences and catalyzes strand exchange, essential for homologous recombination.
In eukaryotic cells, the DNA damage response (DDR) signaling pathway is crucial for regulating and coordinating cellular responses to DNA damage, primarily governed by the ATM (ataxia-telangiectasia mutated) and ATR (ATM- and Rad3-Related) proteins Both ATM and ATR are activated in response to DNA damage and replication stress; however, they exhibit distinct specificities regarding the types of DNA damage they address, and their functions are not interchangeable.
UV radiation is a significant source of DNA damage, causing direct lesions such as cyclobutane pyrimidine dimers (CPDs) and hydrated pyrimidines, as well as 6-4 pyrimidine photoproducts (6-4 PPs) Additionally, it can lead to indirect damage, including double-stranded DNA breaks (DSBs) (Rastogi et al.).
In 2010, it was established that initial photoproducts like cyclobutane pyrimidine dimers (CPDs), 6-4 photoproducts (6-4 PPs), and reactive oxygen species (ROS) are linked to transcription or replication blockage, a phenomenon known as the "collapse of replication forks." This process ultimately results in the formation of double-strand breaks (DSBs), as noted by Limoli et al in 2002 Notably, UV radiation appears to play a minimal role in this context.
DSBs directly but pyrimidine dimers and other photoproducts that lead to replication arrest and DSBs
Double-strand breaks (DSBs) are the most lethal type of DNA lesions, as they impact both strands of DNA, potentially resulting in the loss of genetic material and cell death (Van Gent et al., 2001).
Direct DNA lesions, such as cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4 PPs), can be effectively repaired through a process known as photo-reactivation This mechanism involves the enzyme photolyase, which specifically targets these lesions and utilizes light energy to monomerize the cyclobutane ring, thereby restoring DNA integrity (Essen & Klar, 2006).
Excision repair is a complex, multi-step process that removes modified or damaged bases through base excision repair (BER) and nucleotide excision repair (NER) In the BER pathway, DNA glycosylases eliminate damaged bases, followed by the removal of deoxyribose by a phosphodiesterase Subsequently, RNA polymerase (RNAP) and DNA ligase fill and seal the resulting gap in the DNA strand Conversely, the NER pathway involves multiple steps and protein complexes that detect DNA damage, separate the strands, and assemble the necessary repair machinery to incise the DNA around the lesion The final steps of NER remove the damage-containing oligonucleotide and fill the gap, effectively repairing lesions in one strand of double-stranded DNA.
The "cut-and-patch" mechanism utilizes the undamaged complementary strand as a template to repair damaged strands through two independent pathways: non-homologous end joining (NHEJ) and homologous recombination (HR) (Sonoda et al., 2006) NHEJ directly ligates broken ends and depends on key DNA repair proteins such as Ku70, Ku80, and DNA ligase IV (Chiruvella et al., 2013) However, this repair process is prone to errors, leading to small insertions, deletions, substitutions at the break site, and potential translocations when joining DSBs from different genomic locations (Heidenreich et al., 2003; Lieber, 2010).
The repair of double-strand breaks (DSBs) through homologous recombination (HR) relies on significant sequence homology between the broken DNA strands and the template strands This HR pathway begins with the resection of the double-strand break, which removes the 5' strand and creates a 3' overhang The resulting 3' tail plays a crucial role in the strand invasion process necessary for accurate DNA repair.
The invasion of homologous double-stranded DNA (dsDNA) initiates DNA synthesis, resulting in the formation of a four-stranded DNA structure known as a Holliday junction (HJ) These HJs are dynamic and can undergo branch migration Subsequently, a helicase removes the HJs, producing nicked daughter duplex DNA that is later ligated Unlike non-homologous end joining (NHEJ), homologous recombination (HR) is regarded as an error-free DNA repair mechanism.
Archaeal HR proteins, which are homologs to eukaryotic Rad50 and Mre11, are typically encoded alongside HerA and NurA in a single operon (Constantinesco et al., 2004) In Pyrococcus furiosus, these proteins work together to catalyze the DNA 3’ end resection step (Hopkins & Paull, 2008) The process of strand invasion involves RadA, its paralogs, and SSB, which collectively form Holliday junctions (HJs) Additionally, Hjm helicase and Hjc nuclease are likely responsible for branch migration and resolution of the HJ, respectively (Kvaratskhelia et al., 2001).
In archaeal DNA repair, base excision repair (BER) and homologous recombination (HR) are essential pathways, while eukaryotic-like nucleotide excision repair (NER) appears to be non-functional (She et al., 2017) Notably, in H volcanii, the Rad50-Mre11 complex inhibits HR to prevent excessive chromosome rearrangements, leading to micro-homology-mediated end-joining as the primary mechanism for double-strand break (DSB) repair (Delmas et al., 2009).
1.2.2 The UV response in Sulfolobus is part of the DNA damage response
Crenarchaeal Sulfolobus species thrive in extreme environments characterized by high temperatures and intense UV radiation, which pose significant risks to genome integrity Despite these challenges, their mutation rate remains comparable to that of mesophilic organisms like Escherichia coli This suggests that Sulfolobus has developed highly efficient mechanisms for detecting and repairing DNA damage, ensuring their survival in harsh conditions.
Archaea lack LexA homologs, therefore, also a classical SOS regulon (Eisen & Hanawalt,
In hyperthermophilic archaea, the RadA protein, which is a homolog of RecA, is consistently expressed and shows moderate induction in response to DNA damage Research on the DNA damage response in these organisms highlights the unique expression patterns of RadA, as documented in various studies (Frửls et al., 2007; Gửtz et al., 2007; Lundgren & Bernander, 2007).
MoxR-like protein family
1.3.1 MoxR proteins’ characteristics and cellular functions
MoxR proteins are a distinct clade within the AAA+ ATPase family, which plays a crucial role in protein and DNA remodeling or degradation by utilizing ATP hydrolysis for mechanical work Unlike other AAA+ clades present across all organisms, MoxR has only been identified in bacteria and archaea These proteins frequently co-occur with metal-binding von Willebrand factor A (vWA) domain proteins and are often encoded within the same operon, highlighting their functional relationship Phylogenetic analysis categorizes MoxR proteins into seven subfamilies, with the MRP and RavA subfamilies being the most extensively studied.
MoxR proteins have an AAA+ domain composed of the α-β-α subdomain and the all-α subdomain (Snider & Houry, 2006) The α-β-α core domain harbors the canonical Walker
A and Walker B motifs, sensor 1 and the Arg finger (Snider et al., 2008) The all-α subdomain contains the sensor 2 motif and a conserved Arg interacting with the γ-
The MoxR family of proteins features an α-helical insertion before sensor 2, positioning the all-α subdomain at the rear of the α-β-α core Unlike other AAA+ proteins, this unique arrangement does not create a "lid" over the α-β-α core (Snider et al., 2008; Miller & Enemark, 2016).
MoxR proteins in bacteria function as molecular chaperones, facilitating the maturation of protein complexes like methanol dehydrogenase (MDH) in Paracoccus denitrificans They also play crucial roles in maintaining cell shape, responding to stress, and influencing pathogenicity In Rhizobium leguminosarum, MoxR is essential for cell envelope integrity; its disruption results in abnormal cell sizes and increased membrane sensitivity Additionally, a MoxR protein in Francisella tularensis is vital for tolerance to various stresses and is regulated by the heat shock transcriptional regulator r32, with its mutation impairing the bacterium's infection ability E coli K-12's RavA protein, characterized for its hexamer formation and ATPase activity, interacts with the ViaA protein and modulates the acid stress response via inducible Lysine decarboxylase (Ldcl) This interaction prevents the binding of ppGpp, an inhibitor of Ldcl, and recent findings indicate that RavA and ViaA also regulate the fumarate reductase complex activity.
In the diverse world of archaea, two MoxR proteins have been identified, although this family is prevalent across all archaeal lineages Notably, the p618 protein, a member of the RavA family from the crenarchaeal Acidianus two-tailed virus (ATV), has been shown to interact with the vWA domain protein p892, indicating its significant role in the extracellular tail development of the ATV virion (Scheele et al., 2011) Furthermore, research on another MoxR protein in the euryarchaeon Thermococcus kodakarensis has demonstrated that KOD1 (TkMoxR) can form hexameric and dodecamer structures and has the ability to bind to double-stranded DNA.
The study highlights that among various molecules present with ATP, only the dodecameric MoxR demonstrated the ability to catalyze the decomposition of double-stranded DNA (dsDNA) into single-stranded DNA (ssDNA), suggesting a helicase activity of TkMoxR that may play a role in gene expression regulation (Pham et al., 2014) Despite this finding, the overall functions of MoxR proteins in archaea remain largely unexplored.
1.3.2 The moxR-vWA3 operon in Sulfolobus acidocaldarius
S acidocaldarius genome contains one moxR gene (saci_0976) directly adjacent to a gene encoding for a vWA domain – containing protein (saci_0977) in a predicted operon (Cohen et al., 2016) According to a previous study, Saci_MoxR belongs to the RavA subfamily where a protein such as RavA was extensively studied in E.coli (Wagner, Ph.D dissertation 2017, Snider and Houry 2006; Wong and Houry 2012) The vWA protein encoded in the moxR operon is one of three vWA domain-containing proteins in S acidocaldarius The other two are ArnB, the repressor of the archaellum operon and vWA2 encoded in the same cluster with ArnB but has no effects on the archaellum regulation (Reimann et al., 2012) Therefore, the vWA domain-containing protein associated with MoxR now is named vWA3 A synteny analysis reveals that moxR –vWA operon is highly conserved in Sulfobales and Crenarchaea Around the cluster are genes encoding the single strand binding protein (SSB) and components of a succinate dehydrogenase (SdhABCD) (Cohen et al., 2016)
Scope of the thesis
UV radiation poses a significant threat to the genomic integrity of all living organisms, particularly those in extreme environments, such as hyper-thermophilic archaea Studies on Sulfolobus species exposed to UV irradiation have shown a transcriptional response to DNA damage, notably the upregulation of genes related to the Ups and Ced systems, which are crucial for DNA transport This suggests a novel DNA repair pathway that includes UV-inducible pili and intercellular DNA transfer However, the mechanisms regulating the transcription of genes associated with the Ups and Ced systems, along with other UV-responsive genes, remain largely unexplored Interestingly, while most archaeal transcriptional regulators resemble bacterial ones, the transcription machinery components in archaea are homologous to eukaryotic RNAP II, indicating that archaeal transcription represents a blend of eukaryotic and bacterial characteristics.
During my Ph.D., I focused on uncovering the regulatory mechanisms that influence gene transcription in response to UV irradiation My thesis aims to address key questions related to this topic.
+ What promoter element(s) and regulators are important to transcription of the ups cluster? How are transcriptions of the ups and ced genes regulated in response to UV stress?
+ Within this thesis, I also extended the work in investigating the function of protein MoxR in Sulfolobus that was identified as the interaction partner of UpsX
RESULTS
A conserved hexanucleotide motif is important in UV-inducible promoters in Sulfolobus acidocaldarius
Thuong Ngoc Le, Alexander Wagner, and Sonja-Verena Albers
After UV irradiation, Sulfolobus species utilize a unique DNA repair pathway involving Ups pili and the Ced system Our research identified a crucial DNA motif in the promoters of ups genes and other genes with altered transcription post-UV exposure We employed a β-galactosidase reporter gene assay to analyze various promoter truncations of ups genes, allowing us to pinpoint significant regions within these promoters Furthermore, we introduced point mutations in the target region to investigate the consensus nucleotides that influence UV-induced transcription Ultimately, we discovered a hexanucleotide motif present in the promoters of most UV-up-regulated genes, as well as those down-regulated after UV treatment Mutating this motif in the promoters of upsX and upsE genes produced phenotypes akin to those seen in deletion mutants of the respective genes.
Statement of the own participation
Under the supervision of Sonja-Verena Albers, Thuong Ngoc Le and Alexander Wagner conducted experiments involving the creation of plasmids and mutant strains TNL performed reporter gene assays, qRT-PCR, and microscopy, while AW and TNL carried out bioinformatics analysis and DNA transfer assays The data analysis and manuscript preparation were collaboratively handled by TNL, AW, and SVA.
Effect of UV irradiation on Sulfolobus acidocaldarius and involvement of the general transcription factor TFB3 in early UV response
Frank Schult 1 , Thuong Ngoc Le 2 , Andreas Albersmeier 3 , Bernadette Rauch 1 , Jửrn Kalinowski 3 , Sonja-Verena Albers 2 and Bettina Siebers 1#
1 Molecular Enzyme Technology and Biochemistry (MEB), Biofilm Centre, Centre for Water and Environmental Research (CWE), University of Duisburg-Essen, Universitọtsstr 5, 45141 Essen, Germany
2 Institute of Biology II, Molecular Biology of Archaea, University of Freiburg, Schaenzlestr.1, 79104 Freiburg, Germany
3 Center for Biotechnology (CEBITEC), Universitọt Bielefeld, 33615 Bielefeld, Germany
#Correspondence to Bettina Siebers, bettina.siebers@uni-due.de
Archaea, particularly the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius, face harmful conditions like UV light that can damage DNA Research on the transcription factor TFB3, using a Strep-/FLAG-tagged modified strain tfb3 CSF, showed that UV treatment significantly increases TFB3 protein levels and promoter activity, as evidenced by β-galactosidase assays These findings suggest that TFB3 plays a crucial role in the early UV stress response by inducing its expression while repressing DNA replication and cell cycle progression to facilitate DNA repair Furthermore, the early induction of TFB3 is essential for the subsequent expression of genes related to DNA repair mechanisms, such as those involved in Ups pili formation and the Ced DNA transporter, which are vital for homologous recombination Notably, all these genes are down-regulated in the tfb3 mutant, highlighting the importance of TFB3 in the UV stress response.
Research article 1 Results to the reference strain, confirms the hypothesis that TFB3 acts as an activator of transcription
State of the own participation
In this study, Thuong Ngoc Le (TNL) prepared samples for RNA isolation and transcriptome analysis, conducted qRT-PCR and UV-induced cellular aggregation assays, and analyzed the results TNL also developed the tfb3_HA strain, executed pulldown assays, and interpreted mass spectrometry results This research was conducted at the Molecular Biology of Archaea laboratory at the University of Freiburg, under the guidance of Sonja-Verena Albers.
Archaea, the third domain of life, exhibit a unique lifestyle and physiology that blend traits from both bacteria and eukaryotes Notably, their information processing mechanisms display this mosaic; transcriptional regulators are similar to those in bacteria, while the basal transcription apparatus is a simplified form of the more intricate systems found in eukaryotes.
Archaea possess a more limited set of general transcription factors (GTFs) compared to eukaryotes, featuring a multi-subunit RNA polymerase (RNAP) along with homologues of the eukaryotic TATA box binding protein (TBP) and transcription factor TFIIB (TFB) Notably, many archaeal promoters exhibit structural similarities to the eukaryotic RNA polymerase II system, containing essential sequences such as the TATA box, TFB responsive element (BRE), and initiator element (Inr).
The molecular mechanisms of transcription initiation in archaea involve key players such as TBP, TFB, and RNA polymerase (RNAP) TBP binds to the TATA box, while TFB forms specific interactions with the BRE site, causing DNA to bend and directing transcription In vitro studies confirm that TBP, TFB, and RNAP are sufficient for initiating transcription Additionally, archaea have a homolog of the eukaryotic TFIIE, known as TFE, which enhances transcription processivity, particularly for promoters with weak TBP interactions TFE also aids in promoter escape by competing with the elongation factor Spt4/5 for RNAP binding.
Most sequenced archaeal genomes contain multiple homologs of general transcription factors (GTFs), with variations observed across different species Recent structural analyses have identified homologous relationships among eukaryotic TFIIB, archaeal TFB, and bacterial σ factors The extensive study of GTF multiplicity in the euryarchaeal branch suggests a potential role in helping organisms adapt to fluctuating environmental conditions Specifically, the halophilic euryarchaeon Halobacterium salinarum NRC has been a focus for understanding the function of these multiple GTFs.
The organism is extensively studied and features seven transcription factor B (TFB) proteins and six TATA-binding proteins (TBP), which collaborate in various combinations to regulate gene transcription vital for heat shock response, oxidative stress management, and adaptation to cold temperatures In contrast, the euryarchaeon Pyrococcus furiosus contains two TFBs and one TBP, with research indicating a significant increase in tfb2 transcript levels during heat shock, while tfb1 levels remain constant, highlighting the specific role of TFB2 in high-temperature responses.
Research on crenarchaeota's multiple GTFs is limited compared to the extensive studies on euryarchaeal lineages Model organisms like Sulfolobus acidocaldarius and S solfataricus have three tfb genes and one tbp gene TFBs generally exhibit a conserved structure with two main domains: the N-terminal region, which contains a zinc ribbon motif and a conserved B-finger crucial for RNAP recruitment, and the C-terminal domain, which accounts for two-thirds of the protein and is vital for interacting with the TBP-DNA complex, featuring a helix-turn-helix motif that facilitates sequence-specific binding to the BRE site.
[24] In Sulfolobus species compared to TFB1 and TFB2, TFB3 is significantly truncated It comprises only a short C-terminal domain lacking the B-finger and the HTH motif
The three transcription factor B (TFB) homologs of S acidicaldarius and S solfataricus exhibit distinct domain organizations, with both species encoding TFB1, TFB2, and TFB3 TFB1 and TFB2 are complete proteins, while TFB3 is a truncated variant that lacks the B-finger and flexible linker region, featuring a shortened C-terminal domain without the helix-turn-helix (HTH) motif Key structural elements include zinc-ribbon (Zn), B-finger (B), flexible linker domain (L), and HTH domains.
While TFB1, which supports transcription initiation in vitro [25], functions as a classical TFIIB homolog fulfilling widespread roles in the regulation of housekeeping genes [11,
The alternative transcription factors TFB2 and TFB3 exhibit significantly lower transcript abundance, suggesting they may have specialized functions A genome-wide transcription study of the cell cycle in S acidocaldarius was conducted using microarray technology.
Research article 2 Results experiments showed that tfb2 is induced in the transition from the G1 to S phase, indicating cell cycle-dependent expression and a role in cell cycle regulation [28].
Two independent transcriptome studies using S acidocaldarius and S solfataricus identified tfb3 as one of the most significantly up-regulated genes in response to UV irradiation To investigate the function of TFB3 in UV stress response, Paytubi et al conducted in vitro transcription assays utilizing purified proteins from S solfataricus.
For their analysis, they used DNA fragments comprising promotors that were previously shown to be activated (dps, thsB), repressed (sta1) upon UV treatment or unresponsive to
UV stress (T6, ssb) experiments demonstrated that TFB3 enhances transcription across all tested promoters, necessitating the presence of TFB1 at the promoter site It is believed that TFB3 interacts with the TBP-TFB1-DNA complex to promote RNAP recruitment, thereby acting as a transcriptional activator.
This study investigates the function of TFB3 in the early UV stress response of S acidocaldarius We developed a tfb3 insertion mutant and utilized in vivo tagging for protein immunodetection, along with Co-immunoprecipitation techniques Additionally, we conducted promoter activity studies and a thorough transcriptome analysis to gain insights into TFB3's role.