Molecular mechanism for degradation of transcriptionally stalled RNA polymerase II in the yeast Saccharomyces cerevisiae [Elektronische Ressource] / Eleni Karakasili

Karakasili, Varikas Eleni

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 24
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und
Pharmazie der Ludwig-Maximilians-Universität München








Molecular Mechanism for Degradation of
Transcriptionally Stalled RNA Polymerase II in
the Yeast Saccharomyces cerevisiae




















Eleni Karakasili
from Marousi, Greece


2010



ERKLÄRUNG

Diese Dissertation wurde im Sinne von §13 Abs. 3 bzw. 4 der Promotionsordnung vom
29. Januar 1998 von Herrn Professor Dr. Patrick Cramer betreut.



EHRENWÖRTLICHE VERSICHERUNG

Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.


München, den 19.07.2010




Eleni Karakasili



Dissertation eingereicht am 19.07.2010
1. Gutachter: Prof. Dr. Patrick Cramer
2. Gutachter: Prof. Dr. Dietmar Martin

Mündliche Prüfung am 04.10.2010



II

SUMMARY

Transcription of protein coding genes by RNA polymerase II (RNAPII) is an essential step
in gene expression. Transcription elongation is a highly dynamic and discontinuous
process that includes frequent pausing of RNAPII, backtracking, and arrest both in vitro
and in vivo. Consequently, a multitude of transcription elongation factors are needed for
efficient transcription elongation. When transcription elongation factors fail to “restart”
RNAPII the persistently stalled RNAPII complex prevents transcription and thus has to be
recognized and removed to free the gene for subsequent polymerases. Similarly, DNA
damage causes stalling of RNAPII. In this case, the DNA damage is either repaired by
Transcription-Coupled Repair (TCR) or RNAPII is degraded as a “last resort” mechanism
by the ubiquitin proteasome system. In contrast to RNAPII degradation caused by DNA
damage, the cellular pathway for removal of transcriptionally stalled RNAPII complexes
has remained largely obscure. However, it was speculated that transcriptionally stalled
RNAPII complexes are degraded by the same pathway as RNAPII stalled due to DNA
damage. Here, it is shown that the pathway for degradation of transcriptionally stalled
RNAPII is distinct from the DNA damage-dependent pathway, providing the first
evidence that the cell distinguishes between RNAPII complexes stalled for different
reasons. The novel cellular pathway for transcriptional stalling-dependent degradation of
RNAPII is termed TRADE. Specifically, in the TRADE pathway a different yet
overlapping set of enzymes is responsible for poly- and de-ubiquitylation of
transcriptionally stalled RNAPII. Moreover, the catalytic 20S proteasome is recruited to
transcribed genes indicating that Rpb1 of transcriptionally stalled RNAPII complexes is
degraded at the site of transcription. Importantly, nucleotide starvation and temperature
stress which might mimic natural conditions of transcription elongation impairment also
lead to RNAPII degradation. Finally, this study provides the first evidence that the
mechanism for the controlled degradation of the transcriptionally stalled RNA polymerase
complex might also exist for transcription by RNAPI and RNAPIII. Taken together, the
TRADE pathway elucidated in this study ensures continued transcription.

III

PUBLICATIONS

Parts of the present thesis are submitted for publication:

Karakasili, E. and Sträßer, K. "A novel pathway for degradation of transcriptionally stalled
RNA polymerase II", under revision


A collaboration with the laboratory of Prof. Dr. Patrick Cramer resulted in the following
publication:

Jasiak, A.J., H. Hartmann, Karakasili, E., Kalocsay, M., Flatley, A., Kremmer, E., Sträßer,
K., Martin, D.E., Söding, J., Cramer P. (2008). "Genome-associated RNA polymerase II
includes the dissociable Rpb4/7 subcomplex." J Biol Chem 283 (39): 26423-26427





IV Table of Contents

TABLE OF CONTENTS



ERKLÄRUNG .................................................................................................... II
EHRENWÖRTLICHE VERSICHERUNG ........................... II
SUMMARY ....................................................................................................... III
PUBLICATIONS ............................... IV
TABLE OF CONTENTS ...................................................................................... V
1. INTRODUCTION ........................ 1
1.1. THE CENTRAL DOGMA OF MOLECULAR BIOLOGY ......................................................... 1
1.2. DNA-DEPENDENT RNA POLYMERASES .......................................... 2
1.3. MODIFICATION OF THE CARBOXYL-TERMINAL DOMAIN (CTD) OF RNAPII ................. 3
1.4. THE TRANSCRIPTION CYCLE .......................................................... 5
1.5. TRANSCRIPTION IS A HIGHLY DYNAMIC AND DISCONTINUOUS PROCESS ........................ 7
1.6. TRANSCRIPTION ELONGATION FACTORS ........................................................................ 9
1.6.1. The CTDK-I kinase complex ....... 9
1.6.2. The elongation cleavage factor TFIIS ........................ 10
1.6.3. The THO complex ..................................................... 11
1.6.4. The Bur1/Bur2 kinase complex ................................................................................................. 12
1.7. THE UBIQUITIN-PROTEASOME PATHWAY (UPP) ..........................14
1.7.1. Polyubiquitylation of the substrate ............................ 14
1.7.2. Degradation by the 26S Proteasome ................................................................ 17
1.7.3. Deubiquitylation of the substrate .............................. 18
1.7.4. Non-proteolytic roles of the UPP in transcription .................................... 19
1.8. UBIQUITYLATION AND PROTEASOME-MEDIATED DEGRADATION OF RNAPII UPON DNA
DAMAGE .................................................................................................20
1.9. AIM OF THIS STUDY ......................................25
2. RESULTS ................................................................... 27
2.1 IMPAIRMENT OF TRANSCRIPTION ELONGATION RESULTS IN THE DEGRADATION OF
RPB1, THE LARGEST SUBUNIT OF RNAPII ............................................27
2.1.1 Transcription elongation impairment results in decreased RNAPII occupancy on the gene. .. 27
2.1.2 Transcription elongation impairment results in lower Rpb1 protein levels. ............................. 29
2.1.3 Transcription elongation impairment does not result in the reduction of the protein levels of
other transcription factors. ....................................................................................................................... 31
2.1.4 Depletion of transcription elongation factors also leads to lower Rpb1 levels. ......................... 32
2.1.5 Treatment with the transcription elongation inhibitor 6AU results in lower Rpb1 levels. ...... 33
2.2 REQUIREMENT OF THE CTD OF RPB1 IN THE DEGRADATION OF TRANSCRIPTIONALLY
STALLED RNAPII ...................................................................................................................35
2.3 TEMPERATURE STRESS LEADS TO DEGRADATION OF RNAPII. ........37
V Table of Contents

2.4 TRANSCRIPTIONALLY STALLED RNAPII IS POLYUBIQUITYLATED AND DEGRADED BY THE
UBIQUITIN-PROTEASOME PATHWAY (UPP) ...........................................................................39
2.4.1 Transcriptionally stalled RNAPII is polyubiquitylated .............................. 39
2.4.2 A slower polymerizing form of RNAPII has decreased Rpb1 polyubiquitylation. .................... 41
2.4.3 The 26S proteasome degrades Rpb1 of transcriptionally stalled RNAPII probably at the site of
transcription .............................................................................................................................................. 43
2.5 SUBUNITS OF THE 26S PROTEASOME INTERACT GENETICALLY WITH THE TRANSCRIPTION
ELONGATION FACTORS ...........................................46
2.6 MOLECULAR MECHANISM FOR THE POLYUBIQUITYLATION OF TRANSCRIPTIONALLY
STALLED RNAPII. ..................................................................................50
2.6.1 The polyubiquitin chains on Rpb1 are mainly K63-linked and are required for degradation. 50
2.6.2 The polyubiquitin chain is attached on K330 and K695 of Rpb1. ........... 53
2.6.3 Ubiquitin modifying enzymes involved in polyubiquitylation of Rpb1.................................... 55
2.6.3.1 Ubc4 and Ubc5 are the E2 conjugating enzymes. .............................................. 56
2.6.3.2 Rsp5 but not Elc1 is the E3 ligase. ......................................... 57
2.6.3.3 Investigation of a possible novel E3 ligase............................................................ 59
2.6.3.4 Involvement of the ubiquitylation promoting protein Def1 and the TCR factor Rad26. ..... 60
2.7 MOLECULAR MECHANISM FOR DE-UBIQUITYLATION OF TRANSCRIPTIONALLY STALLED
RNAPII. ....