ARC Centre of Excellence for Advanced Molecular Imaging
Dept. of Biochemistry and Molecular Biology
Building 77, Clayton Campus
23 Innovation Walk
Clayton VIC 3800
We develop methods for single particle electron microscopy (SPEM). Our SPEM developments are motivated by our interest in transcriptional control, because transcription regulatory complexes are among the most difficult SPEM targets. They are asymmetric and their composition and conformation is inherently dynamic as a consequence of their functional roles in regulation of gene expression. Therefore, the structural and biochemical knowledge about higher order transcription regulatory assemblies is fragmental. Our research aims at overcoming this deficit in understanding and methodology to enable high-resolution structure determination of these and other similarly challenging targets for SPEM. The objectives of our SPEM developments are to
(1) remove the requirement for a priori structural knowledge and open the method to the study of particles with novel structure. Robust algorithms for ab initio 3D reconstruction are particularly important for the analysis of small particles with low symmetry.
(2) enable the study of extremely heterogeneous particle populations with SPEM. Macromolecules are dynamic entities that move to exert their function. As we pointed out in our review published in Annual Reviews of Biochemistry current methods for so-called “heterogeneity analysis” are immature and suffer from serious shortcomings. A robust ensemble 3D reconstruction method would open for the study of biological systems currently intractable to structure determination by SPEM.
(3) allow image processing orders of magnitudes faster than currently possible. This will provide the capability for immediate evaluation of data quality and adjustment of data acquisition parameters in real-time. For the researcher undertaking a SPEM investigation, this means increased insight into the nature of the data, improved success rate and enhanced turnover.
(4) eliminate bias due to model, noise and human. The problem of bias is central in SPEM and we must strive to develop approaches capable of overcoming bias of all kinds. We have taken the first steps toward an approach free of bias, but the methods need to be generalised to extremely heterogeneous SPEM image populations and incorporate a more sophisticated image restoration methodology.
Transcription involves reading of the genetic code and production of messenger RNA by RNA polymerase II. Genes are preceded by a DNA sequence called the core promoter, and initiation of transcription requires the assembly of a large number of proteins at the core promoter. This then leads to recruitment of RNA polymerase II and formation of a promoter-bound molecular assembly with ~50 individual subunits called the pre-initiation complex (PIC). PIC formation is the key step for regulating gene expression and represents the end-point of many signalling pathways. Proteins that activate or repress transcription, including nuclear hormone receptors and tumour suppressor proteins, directly act on the PIC. All cellular processes depend on stringent transcription regulation and aberrant transcription is therefore the cause of many human diseases. Our biological aims are to understand how the mechanisms for gene regulation differ between constitutively transcribed (housekeeping) and stress-inducible genes by studying the large but transient protein-protein and protein-DNA complexes involved using a combination of SPEM, mass-spectrometry and biochemical assays.
The eukaryotic genome consists of two classes of genes preceded by distinctly different promoter sequences. Housekeeping genes (90%) code for proteins that need to be produced at all times while stress genes (10%) code for proteins that are produced as responses to stress stimuli, such as starvation or infection. The PIC has a fundamentally different composition on these two classes of genes; 40% of the protein mass is unique in each complex because the one mega Dalton TFIID molecule exclusively regulates housekeeping genes. Most transcription research has focused on stress genes because the reaction is simpler to reconstitute. Biochemical and structural studies describing transcription initiation on housekeeping promoters are non-existent despite their fundamental importance to cellular biology.
Eukaryotic transcriptional co-activators are multi-subunit complexes that both modify chromatin and recognize histone modifications to control gene transcription. SAGA, short for Spt- Ada-Gcn5-Acetyl transferase, is one of the major transcriptional co-activator complexes in eukaryotes. SAGA is conserved from yeast to man and performs multiple functions during transcriptional activation and elongation by RNA polymerase II. A conserved set of TATA-box binding protein (TBP)-associated factors are shared between SAGA and the general transcription factor TFIID. Whilst TFIID regulates housekeeping genes, such as ribosomal protein genes, SAGA is of specific disease relevance as it controls the expression of stress-induced genes activated by key oncogenic transcription factors such as c-Myc. SAGA displays a more complex multi-modular architecture than TFIID, with each module harbouring a distinct functional role or enzymatic activity directly linked to human disease. Mutation or altered expression of SAGA subunits is associated with neurological disease and aggressive cancers in humans. SAGA is recruited to specific target genes by key oncogenic transcription factors to activate and regulate gene transcription. SAGA’s many functions are essential for normal embryo development in flies and mice. However, the paucity of detailed knowledge about the mechanisms that turn on/off the different enzymatic activities of SAGA throughout the transcription cycle has precluded the development of targeted therapies. Several studies have shown that the activities of the enzymatic subunits of SAGA are altered when they are incorporated into larger subcomplexes, underscoring the need for structural information of the complete SAGA assembly to enable structure-guided approaches to inform the design of new therapies.
Dominika & Hans Elmlund