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 significant milestone in evolutionary biology in 1977, extensive research has revealed numerous unique characteristics of archaea, establishing them as a distinct domain It is now well recognized that archaea have 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 complex made up of 13 highly conserved subunits that are homologous to eukaryotic RNAP II The core promoter in archaea typically contains a TATA box, located approximately -26 to -30 bp upstream of the transcription start site (TSS), along with a purine-rich transcription factor B recognition element (BRE) situated directly upstream Additionally, there are less defined elements such as the initiator element (INR) and the promoter proximal element (PPE), though their presence varies among archaeal groups The INR is often less detectable in haloarchaeal promoters but is prominent in methanogens and Sulfolobales, while the PPE is primarily found in Sulfolobus promoters.
Transcription factors are essential for the recruitment of RNAPII and RNAP to promoters, as highlighted by Blombach & Grohmann (2017), Langer et al (1995), and Soppa (1999a) Three key types of transcription factors that play a significant role in the initiation of archaeal transcription have been extensively studied: 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 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) Initially, TFE was found to enhance transcription initiation by improving TATA-box recognition; however, subsequent research revealed its role in stabilizing the transcription bubble during elongation Notably, these three archaeal transcription factors are homologous to eukaryotic TBPs, TFBII, and TFEα, highlighting their evolutionary significance.
& Jackson 2000; Thomas & Chiang 2006; Werner & Weinzierl 2005; Grove 2013)
Archaeal TBPs closely resemble the C-terminal domain of eukaryotic TBPs, sharing a similar function in transcription initiation Comprising approximately 180 amino acids and featuring two direct repeats that are about 40% identical, archaeal TBPs bind to the TATA box to initiate transcription This binding not only bends the promoter but also recruits TFB to the BRE site Recent research has highlighted differences in the TBP-DNA interaction lifetimes between archaeal and eukaryotic systems Eukaryotic interactions follow a linear, two-step bending mechanism, while archaeal TBP bending occurs in a single step, with TFB being essential for this process The stabilization of the TBP-DNA complex by TFB appears to be a specific mechanism that enhances the specificity of archaeal TATA-containing promoters.
TFBs in archaea exhibit notable similarities to eukaryotic TFIIB, characterized by an N-terminal domain of 100-120 amino acids and a C-terminal domain with two repeat sequences of approximately 90 amino acids (Soppa, 1999a) The interaction between TFB and TBP, as well as TFB's binding to the BRE, is facilitated by the C-terminal core domain and the helix-turn-helix (HTH) motif (Bell & Jackson, 2000b; Qureshi et al., 1997; Soppa, 1999b) Recent studies on the crystal structure of TBP and the C-terminal core of TFB (TFBc) from P woesei, in conjunction with a promoter containing a TATA-box and BRE, have revealed intricate stereo-specific interactions between the BRE and the helix-turn-helix motif in TFBc.
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 Notably, 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 archaeal ternary complex, depicted in Figure 1A, illustrates the crystal structure formed by the TATA-binding protein (TBP), the C-terminus of transcription factor B (TFBc), and a TATA-box and BRE-containing oligonucleotide, with DNA represented in pink and yellow, TBP as a green ribbon, and TFBc in magenta (Littlefield et al., 1999; PDB Acc No 1D3U) Figure 1B shows the formation of a pre-initiation complex (PIC) in archaea, where TBP first binds to the TATA box, followed by TFB's interaction with the TBP-DNA complex, ultimately recruiting RNA polymerase (RNAP) and transcription factor E (TFE) Additionally, Figure 1C highlights the role of repressors in regulating transcription by binding to core promoter-overlapping sequences, which hinders the access of basal transcription factors TBP and TFB, as well as RNAP during later stages of PIC assembly Conversely, transcriptional activators enhance PIC assembly by binding to sequences located 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 the initiation phase is the most impactful for controlling gene expression 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 significant role in adjusting gene expression in response to environmental changes Eukaryotes utilize four distinct RNA polymerases and various GTFs for comprehensive transcription regulation Archaeal genomes typically contain at least one TATA-binding protein (TBP) and one transcription factor B (TFB), with some archaea, like Halobacterium NRC-1, having multiple GTF paralogs This particular archaeon has six TBPs and seven TFBs, potentially allowing for 42 unique TFB-TBP combinations to influence transcription initiation Research indicates that Halobacterium NRC-1 employs seven of these pairs, with TBPe being the most frequently interacting TBP In contrast, Methanosarcina acetivorans features three TBP homologs and a single TFB.
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 when acetate concentrations are limited.
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 elements, such as the TATA box and BRE, influences whether the regulator functions as a repressor or 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.
Introduction inhibits the formation of the pre-initiation complex (PIC) by blocking the access of transcription factors such as TBP, TFB, or RNAP to the promoters In contrast, activators typically bind to sequences located upstream of the core promoter Despite this, the number of identified activators in archaea and the understanding of their mechanisms of action remain limited compared to the extensive knowledge available about archaeal repressors.
Transcription regulators and their binding sites on promoter regions are crucial regulatory motifs, primarily serving as binding sites for repressors like the TGM (Thermococcales-glycolytic-motif) in Thermococcales, as well as MDR1, NrpR, and Phr Additionally, some activating motifs, such as ArnR, ss-Lrp, Ptr2, and MalR, have been identified as binding sites for activators Notably, the ARA box, a DNA sequence in the promoter for the arabinose-binding protein (AraS), is recognized as an activating element, though its regulator remains unidentified Most regulatory motifs exhibit a semi-palindromic sequence with variable or non-conserved base pairs at their center, while the binding site of the heat shock regulator Phr in Pyrococcus furiosus is uniquely a non-palindrome.
1.1.2.3 Gene-specific transcriptional regulators: repressors, activators
Archaeal genomes, while having a eukaryotic-like basal transcription machinery, predominantly feature bacterial-like regulators instead of their eukaryotic equivalents Many of these archaeal regulators resemble bacterial helix-turn-helix (HTH) domain proteins, with some exhibiting the ribbon–helix–helix (RHH) motif or the Zn ribbon Notably, transcriptional regulators with an HTH motif typically function as dimers or higher oligomers, binding to palindromic DNA sequences and interacting with two consecutive major groove segments on the same face 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 damage is fully repaired.
The SOS response is a crucial DNA damage response (DDR) mechanism in bacteria, regulated by over 40 independent SOS genes (Janion, 2008; Michel, 2005) Key players in this process are the LexA repressor and the 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, LexA undergoes self-cleavage triggered by the RecA-ssDNA complex, allowing for the expression of SOS genes responsible for DNA repair, division inhibition, and the mutagenic repair polymerase Pol V (Michel, 2005) Additionally, the RecA filament facilitates the invasion of homologous double-stranded DNA sequences and catalyzes strand exchange, which is essential for homologous recombination (Janion, 2008; Kreuzer, 2013; Michel, 2005; Rastogi et al., 2010).
In eukaryotic cells, the DNA damage response (DDR) signaling pathway is crucial for regulating and coordinating cellular reactions to DNA damage, primarily driven by the ATM (ataxia-telangiectasia mutated) and ATR (ATM- and Rad3-Related) proteins While both ATM and ATR are activated in response to DNA damage and replication stress, they exhibit distinct specificities and non-redundant functions.
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 photoproducts (6-4 PPs) Additionally, it can lead to indirect damage, including double-stranded DNA breaks (DSBs) (Rastogi et al.).
In 2010, research highlighted that initial photoproducts like CPDs and 6-4 PPs, along with reactive oxygen species (ROS), can cause transcription or replication blockage, leading to the collapse of replication forks and ultimately resulting in the production of double-strand breaks (DSBs) (Limoli et al., 2002) Notably, UV radiation appears to have a limited role in this process.
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 damage, impacting both strands and potentially resulting in genetic material loss and cell death (Van Gent et al., 2001).
Direct DNA damage, such as cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4 PPs), can be effectively repaired through a process called photoreactivation This mechanism involves the enzyme photolyase, which specifically targets these lesions and utilizes light energy to directly convert the cyclobutane ring back to its monomeric form (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 phosphodiesterase Subsequently, RNA polymerase (RNAP) and DNA ligase work to 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 strands, and assemble the necessary repair machinery to incise the DNA around the lesion The final steps of NER entail the removal of the damage-containing oligonucleotide and the filling of the gap Both NER and BER effectively repair lesions on one strand of double-stranded DNA (dsDNA).
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 repairs double-strand breaks (DSBs) by directly ligating the broken ends, relying on key proteins such as Ku70, Ku80, and DNA ligase IV (Chiruvella et al., 2013) However, this pathway is prone to errors, resulting in small insertions, deletions, substitutions, and translocations when DSBs from different genomic regions are joined (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 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 They are resolved by helicase, which produces nicked daughter duplex DNA that is subsequently ligated Unlike non-homologous end joining (NHEJ), homologous recombination (HR) is recognized as an error-free DNA repair mechanism.
Archaeal HR proteins, including homologs of eukaryotic Rad50 and Mre11, are often found encoded in an operon alongside HerA and NurA In the organism Pyrococcus furiosus, these proteins work together to facilitate the DNA 3’ - end resection step The process of strand invasion is mediated by RadA, its paralogs, and SSB, which contribute to the formation of Holliday junctions (HJs) Additionally, Hjm helicase and Hjc nuclease are likely responsible for branch migration and resolution of the HJ, respectively.
In archaeal DNA repair, Base Excision Repair (BER) and Homologous Recombination (HR) are essential pathways, while eukaryotic-like Nucleotide Excision Repair (NER) does not play a significant role (She et al., 2017) In H volcanii, the Rad50-Mre11 complex inhibits HR-mediated repair of Double-Strand Breaks (DSBs) to prevent excessive chromosome rearrangements, with micro-homology-mediated end-joining serving as the primary mechanism for 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 UV irradiation, which pose significant risks to genome integrity Despite these challenges, their mutation rate is comparable to that of mesophilic organisms like Escherichia coli This suggests that Sulfolobus has developed highly efficient mechanisms for detecting and repairing DNA damage.
Archaea lack LexA homologs, therefore, also a classical SOS regulon (Eisen & Hanawalt,
In hyperthermophilic archaea, the RadA protein, which is homologous to RecA, is consistently expressed and shows moderate induction in response to DNA damage This was highlighted in studies conducted by Frửls et al (2007), Gửtz et al (2007), and Lundgren & Bernander (2007), emphasizing the mechanisms of DNA damage response in these organisms.
MoxR-like protein family
1.3.1 MoxR proteins’ characteristics and cellular functions
MoxR proteins are a distinct group within the AAA+ ATPase family, which plays a crucial role in protein and DNA remodeling or degradation by coupling ATP hydrolysis to mechanical work (Miller & Enemark, 2016; Neuwald et al., 1999; Olivares et al., 2015; Sauer & Baker, 2011) Unlike other AAA+ clades found across various organisms, the MoxR subfamily has only been identified in bacteria and archaea (Iyer et al., 2004) Typically, MoxR proteins are associated with metal-binding von Willebrand factor A (vWA) domain proteins, often encoded within the same operon, suggesting a functional relationship (Snider & Houry, 2006; Wong & Houry, 2012) Phylogenetic analysis categorizes MoxR proteins into seven subfamilies, with the MRP- and RavA-subfamilies being the most extensively studied (Snider & Houry, 2006).
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 proteins feature 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 configuration does not create a "lid" over the top of the α-β-α core (Snider et al., 2008; Miller & Enemark, 2016).
MoxR proteins in bacteria serve as molecular chaperones, facilitating the maturation of protein complexes like methanol dehydrogenase (MDH) in Paracoccus denitrificans They play crucial roles in cell shape maintenance, stress response, and pathogenicity, particularly in Rhizobium leguminosarum, where disruption of moxR results in abnormal cell size and increased membrane sensitivity In Francisella tularensis, a specific MoxR protein is essential for tolerance to various stresses and is regulated by the heat shock transcriptional regulator r32; its mutation significantly impairs the bacterium's infection capability Additionally, the E coli K-12 protein RavA, known for its ATPase activity and hexamer formation, interacts with the ViaA protein and modulates the inducible lysine decarboxylase (Ldcl) for acid stress response, preventing the binding of the alarmone ppGpp that inhibits Ldcl activity Recent findings also indicate that the fumarate reductase complex activity is regulated through the interaction of RavA and ViaA.
In the study of archaea, two MoxR proteins have been identified, despite their prevalence across various archaeal lineages Notably, the protein p618, a member of the RavA family from the crenarchaeal Acidianus two-tailed virus (ATV), interacts with the vWA domain-containing protein p892, indicating its significant role in the extracellular tail development of the ATV virion (Scheele et al., 2011) Additionally, research on another MoxR protein in the euryarchaeon Thermococcus kodakarensis demonstrated that KOD1 (TkMoxR) can form hexameric and dodecamer structures and has the capability to bind to double-stranded DNA.
This study highlights the unique ability of the dodecameric MoxR protein to catalyze the decomposition of double-stranded DNA (dsDNA) into single-stranded DNA (ssDNA) in the presence of ATP, suggesting its helicase activity 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 like hyper-thermophilic archaea Microarray studies on Sulfolobus species after UV exposure demonstrated a transcriptional response to DNA damage, notably the upregulation of genes related to the Ups and Ced systems, which are crucial for DNA transport These findings indicate a potential novel DNA repair pathway involving UV-inducible pili and intercellular DNA transfer However, the mechanisms regulating the transcription of genes associated with the Ups and Ced systems, as well as other UV-responsive genes, remain largely unexplored Interestingly, archaeal transcriptional regulators resemble bacterial ones, while their transcription machinery components are homologous to eukaryotic RNAP II, suggesting that archaeal transcription is a hybrid of eukaryotic and bacterial features.
During my Ph.D., I investigated the regulatory mechanisms that control gene transcription in response to UV irradiation The focus of my thesis is to address key questions regarding this process.
+ 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
Following UV irradiation, Sulfolobus species utilize a unique DNA repair pathway involving Ups pili and the Ced system This study identifies a crucial DNA motif in the promoters of ups genes and other genes with altered transcription post-UV exposure Using a β-galactosidase reporter gene assay, we screened various truncations of ups gene promoters to pinpoint essential regions Additionally, we created point mutations in the target region to analyze the consensus nucleotides that influence UV-induced transcription Our findings revealed a hexanucleotide motif present in the promoters of most UV-upregulated genes and those that were downregulated after UV treatment Notably, mutating this motif in the promoters of upsX and upsE genes produced phenotypes similar to those seen in deletion mutants of these genes.
Statement of the own participation
Under the supervision of Sonja-Verena Albers, Thuong Ngoc Le (TNL) and Alexander Wagner (AW) conducted experiments involving the creation of plasmids, mutant strains, and various assays, including reporter gene assays, qRT-PCR, and microscopy Both TNL and AW were responsible for bioinformatics analysis and DNA transfer assays The collaborative efforts of TNL, AW, and SVA culminated in data analysis and manuscript preparation.
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 cause DNA damage Research focused on the alternative general transcription factor TFB3, which is crucial for the UV stress response Using a modified strain with Strep-/FLAG-tagged TFB3, we observed an increase in TFB3 protein levels and promoter activity following UV exposure These findings suggest that the initial responses to UV irradiation involve the induction of TFB3 and repression of replication and cell cycle progression to facilitate DNA repair The early activation of TFB3 is essential for the subsequent expression of genes related to Ups pili formation and the Ced DNA transporter, both of which are vital for DNA transfer and homologous recombination repair Notably, all 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) conducted RNA isolation and transcriptome analysis, utilizing qRT-PCR and UV-induced cellular aggregation assays TNL also developed the tfb3_HA strain, executed the pulldown assay, and analyzed mass spectrometry (MS) results This research was carried out at the Molecular Biology of Archaea laboratory at the University of Freiburg, under the guidance of Sonja-Verena Albers.
Archaea exhibit a unique lifestyle and physiology that combines features of both bacteria and eukaryotes, particularly in information processing Their transcriptional regulators are more similar to those of bacteria, while their basal transcription apparatus is a simplified version of the complex system seen in eukaryotes.
Archaea possess a more limited set of general transcription factors (GTFs) compared to eukaryotes, featuring a multi-subunit RNA polymerase (RNAP) and homologues of the eukaryotic TATA box binding protein (TBP) and transcription factor TFIIB (TFB) The structural characteristics of most archaeal promoters bear resemblance to the eukaryotic RNA polymerase II system, as they include essential elements 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 TBP binds to the TATA box, while TFB forms specific interactions with the BRE site, causing DNA bending that directs transcription In vitro studies indicate that TBP, TFB, and RNA polymerase are sufficient for initiating transcription Additionally, archaea have a homolog of eukaryotic TFIIE, known as TFE, which enhances transcription processivity by boosting the activity of certain promoters, particularly those with weak TBP interactions TFE also facilitates promoter escape by competing with the elongation factor Spt4/5 for RNA polymerase binding.
Most sequenced archaeal genomes contain multiple homologs of general transcription factors (GTFs), with variation across species Recent structural analyses indicate that eukaryotic TFIIB, archaeal TFB, and bacterial σ factors share homology The diversity of GTFs has been extensively researched within the euryarchaeal branch, suggesting a role in adapting to fluctuating environmental conditions for various organisms Notably, the halophilic euryarchaeon Halobacterium salinarum NRC has been highlighted in studies regarding the function of these multiple GTFs.
The organism studied is highly researched and features seven transcription factor B (TFB) proteins and six TATA-binding proteins (TBP), which work together to regulate gene transcription crucial for heat shock response, oxidative stress, and adaptation to low temperatures In contrast, the euryarchaeon Pyrococcus furiosus contains two TFBs and one TBP, with studies indicating that the transcript levels of tfb2 significantly increase during heat shock, while tfb1 levels remain constant, highlighting the potential role of TFB2 in high-temperature responses.
Research on crenarchaeota's GTFs is limited compared to the well-studied euryarchaeal lineage The thermoacidophilic model organisms Sulfolobus acidocaldarius and S solfataricus each contain three tfb genes and one tbp gene TFBs exhibit a conserved structure with two primary domains: the N-terminal domain features a zinc ribbon motif and a conserved B-finger, crucial for RNAP recruitment, while the C-terminal domain, which constitutes two-thirds of the protein, is vital for interacting with the TBP-DNA complex and contains a helix-turn-helix (HTH) motif for 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 domain organization of the three transcription factor B (TFB) homologs in Sulfolobus acidocaldarius and Sulfolobus solfataricus reveals that both species possess three homologs of the eukaryotic general transcription factor TFIIB (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 components include zinc-ribbon (Zn), B-finger (B), flexible linker domain (L), and HTH domain.
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 roles A genome-wide transcription analysis 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 on S acidocaldarius and S solfataricus identified tfb3 as a significantly up-regulated gene in response to UV irradiation To investigate the function of TFB3 in UV stress response, Paytubi et al conducted in vitro transcription assays using 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) enhances transcription from various promoters, with TFB3 playing a crucial role The presence of TFB1 at the promoter is essential for this process, suggesting that TFB3 interacts with the TBP-TFB1-DNA complex to facilitate RNAP recruitment, thereby acting as a transcriptional activator.
This study investigates the function of TFB3 in the early UV stress response of S acidocaldarius through the development of a tfb3 insertion mutant, in vivo tagging for protein immunodetection, Co-immunoprecipitation, promoter activity assessments, and an extensive transcriptome analysis.