Molecular characterization of factors involved in regulation of archaeal translation

F. Blombach

Research output: Thesisinternal PhD, WU

Abstract

The three domains of life – Bacteria, Archaea, and Eukaryotes – can be easily distinguished based on how the genetic information is processed during transcription, translation, and (DNA) replication. Generally, Eukaryotes turned out to employ machineries for these processes that are in their essence homologous to the corresponding archaeal machineries. This prompted the idea of an “archaeal parent” at the beginning of eukaryotic evolution.
This thesis deals with various aspects of translation in the crenarchaeon Sulfolobus solfataricus and Archaea in general. Numerous proteins are directly involved in the translation process as factors regulating initiation, elongation, and termination, or as ribosomal proteins being an integral part of the ribosome, the machinery catalyzing protein synthesis. These proteins usually show a high level of conservation and a considerable part of these proteins is common to all cellular life. Other proteins aiding the translation machinery can be involved in different translation-related processes. This starts with the assembly of the ribosomal subunits from rRNA molecules and ribosomal proteins. The synthesis of ribosomal RNA by an RNA polymerase is the starting point of ribosome assembly. Eukaryotes possess RNA polymerases I and III that are specially dedicated to the transcription of ribosomal RNA and other structural RNAs, i.e. RNA molecules that are not translated into proteins but serve for instance as building blocks for the translation machinery. Archaea and Bacteria are thought to have only a single type of RNA polymerase.
Chapter 6 of this thesis describes the identification of an archaeal ortholog of RNA polymerase III subunit RPC34. This subunit was considered to be unique for eukaryotic RNA polymerase III and plays an essential role in the recruitment of the RNA polymerase to promoter-bound general transcription factors. The archaeal RPC34 ortholog is predicted to function in transcription as well. Gene synteny analysis revealed that the archaeal gene orthologs cluster with various genes involved in the modification and processing of structural RNAs. The functional separation of transcription of protein-encoding and structural RNAs in Eukaryotes might therefore date back to the “archaeal parent” of Eukaryotes, with RPC34 playing a key role therein.
As stated above, the stepwise assembly of the ribosome begins already during the transcription of rRNA, with the first ribosomal proteins binding to the growing rRNA transcript. This process is controlled by a multitude of factors and in Bacteria and Eukaryotes a major group of ribosome assembly factors are GTPases of the translation-factor related class (TRAFAC). There are only a few TRAFAC GTPases in Archaea that serve as candidates for a role in ribosome assembly, as outlined in Chapter 1. In Chapter 2, one of those archaeal TRAFAC GTPases, the HflX ortholog SsGBP from S. solfataricus was studied in structural detail. The crystal structure revealed a two-domain arrangement including a prototypic HflX-domain that probably functions as a nucleic acid binding domain. The presence of the HflX-domain influences the GTP-hydrolysing properties of the C-terminal G-domain. Overall, the enzyme shows biochemical properties similar to other translational GTPases with slow intrinsic GTPase activity and relatively low affinity for GTP.
Chapter 3 describes the interaction of SsGBP with the large ribosomal subunit. Binding
assays show that SsGBP similar to its bacterial orthologs binds to the 50S ribosomal subunit both in GDP- and GTP-bound form. Apo-SsGBP shows less stable binding. These results were somewhat unexpected as no conformational changes were observed between the GDP-bound and apo-SsGBP crystal structures whereas GTP-binding was predicted to drive major conformational rearrangements (Chapter 2). In line with the prediction derived from the SsGBP crystal structures, the HflX domain provides a major surface for the ribosome interaction of SsGBP.
In S. solfataricus the gene coding for SsGBP is co-transcribed with a gene coding for an archaeal ortholog of the eukaryotic transcription co-activator MBF1. Archaeal and eukaryotic MBF1 orthologs share a conserved helix-turn-helix domain. Chapter 4 describes the results of a genetic approach for the characterization of the mbf1 gene. It is shown that, at least under the conditions tested, mbf1 is not essential in the crenarchaeon Sulfolobus solfataricus and gene deletion caused only a mild phenotype and little change on transcriptome level. In Chapter 5 it is shown that the archaeal MBF1 shows properties that differ from the published data for its eukaryotic ortholog. While no evidence was found for an interaction with the transcription machinery, the archaeal MBF1 ortholog does bind to ribosomes engaged in translation via the 30S ribosomal subunit. Intriguingly, the helix-turn-helix domain of archaeal MBF1 provides the major binding surface for the interaction with the 30S ribosomal subunit.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • van der Oost, John, Promotor
  • Brouns, Stan, Co-promotor
Award date19 May 2010
Place of Publication[S.l.
Publisher
Print ISBNs9789085856436
Publication statusPublished - 2010

Keywords

  • ribosomes
  • translation
  • gene expression
  • bacteria
  • transcription
  • rna polymerase
  • archaea
  • regulation of transcription

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