Regulation of the Ups pili system involved in DNA damage response in Sulfolobus

Regulation of the Ups pili system involved in

DNA damage response in Sulfolobus

Inaugural-Dissertation

zur

Erlangung des Doktortorwürde der Naturwissenschaften (Dr. rer. nat.) am

Fachbereich Biologie der Albert-Ludwigs-Universität

Freiburg im Breisgau

Thuong Ngoc Le

geboren am 19.08.1988 in Thai Nguyen, Vietnam.

Regulation of the Ups pili system involved in

DNA damage response in Sulfolobus

Inaugural-Dissertation

zur

Erlangung des Doktortorwürde der Naturwissenschaften (Dr. rer. nat.) am

Fachbereich Biologie der Albert-Ludwigs-Universität

Freiburg im Breisgau

Thuong Ngoc Le

geboren am 19.08.1988 in Thai Nguyen, Vietnam.

The light microscopy picture on the cover shows S. acidocaldarius cells forming aggregates due to

UV irradiation. The picture was taken by Thuong Ngoc Le.

Die vorliegende Arbeit wurde von Febuary 2014 bis August 2014 am Max-Planck Institut

für terrestrische Mikrobiologie in Marburg und von September 2014 bis Marz 2018 an der

Albert-Ludwigs-Universität Freiburg in der Arbeitsgruppe von Frau Prof. Dr. Sonja-Verena

Albers durchgeführt.

Dekanin der Fakultät für Biologie: Prof. Dr. Bettina Warscheid

Promotionsvorsitzender: Prof. Dr. Andreas Hiltbrunner

Betreuer der Arbeit:

Referent: Prof. Dr. Sonja-Verena Albers

Koreferent:

Drittprüfer:

Datum der mündlichen Prüfung:

The results that I have achieved during my Ph.D., which are described in this thesis, are

published or to be published in the following peer review articles:

1. Thuong Ngoc Le, Alexander Wagner and Sonja-Verena Albers. A conserved

hexanucleotide motif is important in UV-inducible promoters in Sulfolobus acidocaldarius.

Microbiology 2017;163: 778-788

2. Frank Schult1

, Thuong Ngoc Le2

, Andreas Albersmeier3

, Bernadette Rauch1

, Jörn

Kalinowski3

, Sonja-Verena Albers2 and Bettina Siebers1#. Effect of UV irradiation on

Sulfolobus acidocaldarius and involvement of the general transcription factor TFB3 in

early UV response

Molecular Microbiology (in submission)

Table of contents

1. INTRODUCTION ..................................................................................... 1

1.1. Transcription in archaea: a mosaic of eukaryotic and bacterial features ................ 2

1.1.1. Basal transcriptional machinery in archaea ................................................................... 2

1.1.2. Regulation of transcription in archaea ........................................................................... 5

1.1.2.1. Regulation of transcriptional initiation by general transcription factors ................. 5

1.1.2.2. Regulatory motifs in archaeal promoters................................................................. 5

1.1.2.3. Gene-specific transcriptional regulators: repressors, activators.............................. 6

1.1.2.4. The role of chromatin binding proteins in transcription regulation......................... 7

1.1.2.5. The regulatory role of non-coding RNAs (ncRNAs) in gene expression.................... 8

1.2. The DNA damage response in hyper-thermophilic archaea ............................................. 9

1.2.1. The DNA damage response (DDR) .................................................................................. 9

1.2.1.1. UV - induced DNA damages...................................................................................... 9

1.2.1.2. DNA repair mechanisms.......................................................................................... 10

1.2.2. The UV response in Sulfolobus is part of the DNA damage response......................... 11

1.2.2.1. The hyper-thermophilic Sulfolobus......................................................................... 11

1.2.2.2. The Ups system in Sulfolobus.................................................................................. 12

1.2.2.3. The Ced system ....................................................................................................... 14

1.2.3. Regulation of the Ups and Ced system in Sulfolobus................................................... 14

1.2.3.1. Transcription factor B3 (TFB3) ................................................................................ 14

1.2.3.2. Other players might be involved in regulation of the UV response in Sulfolobus.. 15

1.3. MoxR-like protein family ............................................................................................. 16

1.3.1. MoxR proteins’ characteristics and cellular functions................................................. 16

1.3.2. The moxR-vWA3 operon in Sulfolobus acidocaldarius................................................ 18

1.4. Scope of the thesis....................................................................................................... 19

2. RESULTS............................................................................................... 20

Research article 1............................................................................................................... 21

A conserved hexanucleotide motif is important in UV-inducible promoters in Sulfolobus

acidocaldarius.................................................................................................................... 21

Research article 2 ............................................................................................................... 37

Effect of UV irradiation on Sulfolobus acidocaldarius and involvement of the general

transcription factor TFB3 in early UV response ....................................................................37

Research article 3 ............................................................................................................... 72

Characterization of a MoxR AAA+ ATPase in Sulfolobus acidocaldarius................................ 72

3. DISCUSSION..........................................................................................97

3.1. The role of the transcription factor TFB3 in the transcriptional regulatory network in

response to UV-induced damage DNA in Sulfolobus ............................................................ 99

3.2. Transcriptional regulators and chromatin-binding proteins: their interplay and

evolutionary relationship ................................................................................................. 103

4. Thesis summary .................................................................................. 108

Zusammenfassung............................................................................................................ 109

References.............................................................................................. 110

Acknowledgment.................................................................................... 116

Introduction

1

1. INTRODUCTION

Introduction

2

1.1. 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,

1977). Since that milestone of evolution biology, a great number of studies have unveiled

more unique characteristics of archaea that make them a separated domain (White,

2006; Cavicchioli, 2010; Werner & Grohmann, 2011; Albers & Meyer, 2011; Lindås &

Bernander, 2013; Karr et al., 2017). Nowadays, it is well established that archaea possess

a transcription apparatus resembling a simplified version of the eukaryotic RNA

polymerase (RNAP) II system (Soppa 1999; Geiduschek & Ouhammouch 2005; Grohmann

& Werner 2011; Orell et al. 2013; Karr 2014; Gindner et al. 2014; Kessler et al. 2015).

Studies on archaeal RNAP from Sulfolobus acidocaldarius delivered the first hint that

archaea might initiate transcription in a eukaryotic manner (Zillig et al., 1979). Archaeal

RNAP is a protein complex composed of 13 subunits of which each individual subunit is

highly conserved and homologous to that of eukaryotic RNAP II (Langer et al., 1995;

Grohmann et al., 2009). Subsequently, the canonical core promoter of archaea was

shown to harbor a TATA box, an AT-rich region located around -26 to -30 bp upstream of

the transcription start site (TSS). Directly upstream of the TATA box is a purine-rich

segment named transcription factor B recognition element (BRE) (Soppa 1999; Qureshi et

al. 1997). There are other less defined DNA elements in archaeal promoters, such as the

initiator element (INR), and the promoter proximal element (PPE) (Peng et al., 2009a;

Soppa, 1999b). However, the presence of the INR and PPE varies among different groups

of archaea. For example, the INR positioned within the initially transcribed region is

hardly detectable in haloarchaeal promoters, but very pronounced in methanogens and

Sulfolobales (Soppa, 1999b). The PPE located between the TATA box and the TSS has

been found primarily in Sulfolobus promoters (Peng et al., 2009a, 2011; Wurtzel et al., 2010).

Neither RNAPII nor RNAP can be recruited to promoters without the aid of transcription

factors (Blombach & Grohmann, 2017; Langer et al., 1995; Soppa, 1999a). So far, three

types of transcription factors involving initiation of archaeal transcription have been well

studied. They are TATA-binding proteins (TBPs), transcription factor B (TFBs), and

Introduction

3

transcription factor E (TFEs). Out of the three, TBP and TFB are necessary and sufficient

for promoter-specific transcription in vitro (Bell et al., 1998). Assembly of TBP and TFB on

the promoter recruits RNAP to the TSS and consequently forms the pre-initiation complex

(PIC). TFE was initially shown to facilitate transcription initiation by enhancing TATA-box

recognition (Bell et al., 2001). Another study, however, demonstrated that TFE stabilizes

the transcription bubble during elongation (Grünberg et al., 2007). These three archaeal

transcription factors are homologs of eukaryotic TBPs, TFBII, and TFEα, respectively (Bell

& Jackson 2000; Thomas & Chiang 2006; Werner & Weinzierl 2005; Grove 2013).

Archaeal TBPs are almost identical to the C-terminal domain of TBPs in eukaryotes

(Soppa, 1999a, 2001). Additionally, their function is also equivalent to the role of

eukaryotic TBPs. Archaeal TBPs are about 180 amino acids long and consist of two direct

repeats that are around 40% identical to each other (Soppa, 1999a). Transcription

initiates by the recognition and binding of TBP to the TATA box. Moreover, binding of TBP

helps to bend the promoter and recruits TFB to the BRE site (Bell et al., 1999a; Qureshi et

al., 1997; Soppa, 2001). Recently, a study revealed the differences of the lifetime of the

TBP– DNA interaction between the archaeal and eukaryotic system (Gietl et al., 2014). For

instance, the eukaryotic DNA-TBP interaction follows a linear, two step-bending

mechanism with an intermediate state having a distinct bending angle. Here, TFBII helps

to stabilize the fully bent TBP– promoter DNA complex. On the other hand, the bending of

the archaeal promoter by TBP is a single step and TFB is strictly required for that process.

However, stabilization of the TBP–DNA complex by TFB does not seem to be a general

mechanism, but probably an additional mechanism that mediates specificity among

archaeal TATA-containing promoters (Gietl et al., 2014).

TFBs in archaea show significant similarity to eukaryotic TFIIB. The protein consists of an N￾terminal domain of 100-120 amino acids and a C-terminal domain containing two repeat

sequences of around 90 amino acids (Soppa, 1999a). The C-terminal core domain and the

helix-turn-helix (HTH) motif of TFB are responsible for interaction of TFB with TBP and binding

of TFB to the BRE, respectively (Bell & Jackson, 2000b; Qureshi et al., 1997; Soppa, 1999b).

The crystal structure of TBP and the C-terminal core of TFB (TFBc) from P. woesei in a complex

with a promoter containing TATA-box and BRE provided a detailed picture of the stereo

specific interactions between the BRE and a helix–turn–helix motif in the C-terminal of TFBc

Introduction

4

(Littlefield et al., 1999). More importantly, the interaction of TFB with TBP-DNA serves as a

platform to recruit RNAP to the TSS (Nikos & Christos, 1999; Soppa, 1999a). The N-terminal

domain of TFB harbors a zinc (Zn) ribbon motif that interacts with RNAP (Soppa, 1999a). In

contrast to eukaryotes, opening of the archaeal promoter does not require energy (Hausner

& 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).

Figure 1: A: Crystal structure of the archaeal ternary complex formed between TBP, TFBc (C-terminus of

TFB) and a TATA-box and BRE – containing oligonucleotide. Molecules are coded in colors, pink and yellow:

DNA; green ribbon: TBP; magenta: TFBc (Littlefield et al., 1999); PDB Acc. No. 1D3U. B: Formation of a pre￾initiation complex (PIC) in archaea. Initially, TATA-binding protein (TBP) recognizes and binds to the TATA

box, the transcription factor B (TFB) interacts with the TBP– DNA complex and the DNA–TBP–TFB complex

subsequently recruits RNAP and transcription factor E (TFE). C: Repressors regulate transcription by binding

to core promoter-overlapping sequences, thereby preventing access of the basal transcription factors TBP

and TFB to promoters or of RNAP in a later stage of PIC assembly. On the other hand, transcriptional

activators function in the initial steps of PIC assembly by binding to the sequences upstream of the BRE.

Figures B, C were adapted from (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