Expected Preparations:

  Cell cycle:
Replication control and mechanism; Phases of the cell-cycle; Checkpoints and apoptosis.
  
  If you are not already familiar with the prior knowledge listed above, you need to prepare yourself from other information sources.  
Keywords: Yeast cell cycle review
Objectives:

This unit will …

  • Review facts about the cell cycle, from the perspective of the saccaromyces cerevisiae model organism;

  • Discuss some issues about organizing these facts;

  • Introduce the yeast Mbp1 protein, as one of the key players .

 Outcomes:

After working through this unit you should…

  • be familiar with the key aspects of the cell cycle and recognize the terminology when you encounter it elsewhere;

  • be able to sketch the phases of the cell cycle;

  • recognize the names of the key players in this biological system and recognize their roles;

  • have a sense of the scope of information that is available about Mbp1 in public databases;

  • recognize the terminology for the functional components of Mbp1 when you encounter such concepts in later units and elsewhere.

Deliverables:

Time management: Before you begin, estimate how long it will take you to complete this unit. Then, record in your course journal: the number of hours you estimated, the number of hours you worked on the unit, and the amount of time that passed between start and completion of this unit.

Journal: Document your progress in your Course Journal. Some tasks may ask you to include specific items in your journal. Don’t overlook these.

Insights: If you find something particularly noteworthy about this unit, make a note in your insights! page.

Evaluation:

NA: This unit is not evaluated for course marks.

Contents

This unit reviews facts about the cell cycle, introduces its key players in yeast, and discusses issues about organizing these facts. It also introduces the yeast Mbp1 transcription factor that we will be using as an example for bioinformatic analysis in later units.

The cell cycle is a biological process that is universally conserved, since all life (as we know it) is based on the replication of cells. It has been intensely studied in all mnners of organisms, yet our understanding is certainly not yet complete. Budding yeast in particular has been a pructive model organism for cell cycle studies and you have learned about the principles in earlier courses, or even in high school. But since so much data is available, this makes thie cell cycle systems good candidates to study the various aspects in the units of this knowledge network.

Let’s start with a general overview and then consider one protein in particular, the Mbp1 transcription factor, in more detail.

 

Task…

  • Read the introductory notes on cell cycle conceptsPDF.
  • Download the following three papers and review them. You should have an understanding of the general layout of the cycle, and the players involved in the G1/S transition.

Travesa, Anna et al.. (2013). “Repression of G1/S transcription is mediated via interaction of the GTB motifs of Nrm1 and Whi5 with Swi6”. Molecular and Cellular Biology 33(8):1476–86 .
[PMID: 23382076] [DOI: 10.1128/MCB.01333-12]

In Saccharomyces cerevisiae, G1/S transcription factors MBF and SBF regulate a large family of genes important for entry to the cell cycle and DNA replication and repair. Their regulation is crucial for cell viability, and it is conserved throughout evolution. MBF and SBF consist of a common component, Swi6, and a DNA-specific binding protein, Mbp1 and Swi4, respectively. Transcriptional repressors bind to and regulate the activity of both transcription factors. Whi5 binds to SBF and represses its activity at the beginning of the G1 phase to prevent early activation. Nrm1 binds to MBF to repress transcription as cells progress through S phase. Here, we describe a protein motif, the GTB motif (for G1/S transcription factor binding), in Nrm1 and Whi5 that is required to bind to the transcription factors. We also identify a region of the carboxy terminus of Swi6 that is required for Nrm1 and Whi5 binding to their target transcription factors and show that mutation of this region overrides the repression of MBF- and SBF-regulated genes by Nrm1 and Whi5. Finally, we show that the GTB motif is the core of a functional module that is necessary and sufficient for targeting of the transcription factors by their cognate repressors.

McInerny, Christopher J. (2011). “Cell cycle regulated gene expression in yeasts”. Advances in Genetics 73:51–85 .
[PMID: 21310294] [DOI: 10.1016/B978-0-12-380860-8.00002-1]

The regulation of gene expression through the mitotic cell cycle, so that genes are transcribed at particular cell cycle times, is widespread among eukaryotes. In some cases, it appears to be important for control mechanisms, as deregulated expression results in uncontrolled cell divisions, which can cause cell death, disease, and malignancy. In this review, I describe the current understanding of such regulated gene expression in two established simple eukaryotic model organisms, the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. In these two yeasts, the global pattern of cell cycle gene expression has been well described, and most of the transcription factors that control the various waves of gene expression, and how they are in turn themselves regulated, have been characterized. As related mechanisms occur in all other eukaryotes, including humans, yeasts offer an excellent paradigm to understand this important molecular process.

Haase, Steven B and Curt Wittenberg. (2014). “Topology and control of the cell-cycle-regulated transcriptional circuitry”. Genetics 196(1):65–90 .
[PMID: 24395825] [DOI: 10.1534/genetics.113.152595]

Nearly 20% of the budding yeast genome is transcribed periodically during the cell division cycle. The precise temporal execution of this large transcriptional program is controlled by a large interacting network of transcriptional regulators, kinases, and ubiquitin ligases. Historically, this network has been viewed as a collection of four coregulated gene clusters that are associated with each phase of the cell cycle. Although the broad outlines of these gene clusters were described nearly 20 years ago, new technologies have enabled major advances in our understanding of the genes comprising those clusters, their regulation, and the complex regulatory interplay between clusters. More recently, advances are being made in understanding the roles of chromatin in the control of the transcriptional program. We are also beginning to discover important regulatory interactions between the cell-cycle transcriptional program and other cell-cycle regulatory mechanisms such as checkpoints and metabolic networks. Here we review recent advances and contemporary models of the transcriptional network and consider these models in the context of eukaryotic cell-cycle controls.

 

One key player in the G1/S cell-cycle switch is the Mbp1 protein, one of the MBF components. It is a transcription factor with a characteristic DNA-binding domain (the APSES, or KilA-N domain), and other notable features and interaction domains. The 3-D structure of the DNA-binding domain is known. We will develop our methods of sequence analysis by using this protein as an example.

Task…

Review the following information items for an introducion to the yeast Mbp1 protein.

Mbp1 at the Saccharomyces Genome Database
Incidentally: what is this information resource?

Mbp1 at WikiGenes
Same question as above: what is this information resource?

Taylor, I A et al.. (2000). “Characterization of the DNA-binding domains from the yeast cell-cycle transcription factors Mbp1 and Swi4”. Biochemistry 39(14):3943–54 .
[PMID: 10747782] [DOI: 10.1021/bi992212i]

The minimal DNA-binding domains of the Saccharomyces cerevisiae transcription factors Mbp1 and Swi4 have been identified and their DNA binding properties have been investigated by a combination of methods. An approximately 100 residue region of sequence homology at the N-termini of Mbp1 and Swi4 is necessary but not sufficient for full DNA binding activity. Unexpectedly, nonconserved residues C-terminal to the core domain are essential for DNA binding. Proteolysis of Mbp1 and Swi4 DNA-protein complexes has revealed the extent of these sequences, and C-terminally extended molecules with substantially enhanced DNA binding activity compared to the core domains alone have been produced. The extended Mbp1 and Swi4 proteins bind to their cognate sites with similar affinity [K(A) approximately (1-4) x 10(6) M(-)(1)] and with a 1:1 stoichiometry. However, alanine substitution of two lysine residues (116 and 122) within the C-terminal extension (tail) of Mbp1 considerably reduces the apparent affinity for an MCB (MluI cell-cycle box) containing oligonucleotide. Both Mbp1 and Swi4 are specific for their cognate sites with respect to nonspecific DNA but exhibit similar affinities for the SCB (Swi4/Swi6 cell-cycle box) and MCB consensus elements. Circular dichroism and (1)H NMR spectroscopy reveal that complex formation results in substantial perturbations of base stacking interactions upon DNA binding. These are localized to a central 5’-d(C-A/G-CG)-3’ region common to both MCB and SCB sequences consistent with the observed pattern of specificity. Changes in the backbone amide proton and nitrogen chemical shifts upon DNA binding have enabled us to experimentally define a DNA-binding surface on the core N-terminal domain of Mbp1 that is associated with a putative winged helix-turn-helix motif. Furthermore, significant chemical shift differences occur within the C-terminal tail of Mbp1, supporting the notion of two structurally distinct DNA-binding regions within these proteins.

Deleeuw, Lynn, Anna V Tchernatynskaia, and Andrew N Lane. (2008). “Thermodynamics and specificity of the Mbp1-DNA interaction”. Biochemistry 47(24):6378–85 .
[PMID: 18491920] [DOI: 10.1021/bi702339q]

The DNA binding domain of the yeast transcription factor Mbp1 is a winged helix-turn-helix structure, with an extended DNA binding site involving C-terminal “tail” residues. The thermodynamics of the interaction of the DNA binding domain with its target DNA sequence have been determined using fluorescence anisotropy and calorimetry. The dissociation constant was determined as a function of pH and ionic strength in assessing the relative importance of specific and nonspecific ionic interactions. Mutational analysis of the residues in the binding site was used to determine their contributions to binding. The three tail histidine residues and His 63 in the recognition helix accounted for most of the pH dependence of the DNA binding. The tail histidine residues, along with two previously identified lysine residues, account for a major part of the polyelectrolyte contribution to binding and for the nonspecific affinity of Mbp1 for DNA. Gln67 was shown to be a very important residue, which interacts in the minor groove of the target DNA. Systematic mutations of the DNA consensus binding sites showed that the CGCG core contributes most to recognition. Isothermal titration calorimetry revealed a strong temperature-dependent enthalpy change, with a Delta Cp of -1.3kJ mol(-1) K(-1), consistent with a specific binding mode and burial of surface area. Parsing the free energy contributions demonstrates that polyelectrolyte effects account for half of the total free energy at the physiological pH and salt concentration. We present a model for the origin of the sequence specificity and overall affinity of the protein that accounts for the observed thermodynamics.

 

Questions, comments

If in doubt, ask! If anything about this contents is not clear to you, do not proceed but ask for clarification. If you have ideas about how to make this material better, let’s hear them. We are aiming to compile a list of FAQs for all learning units, and your contributions will count towards your participation marks.

Improve this page! If you have questions or comments, please post them on the Quercus Discussion board with a subject line that includes the name of the unit.

References

Page ID: FND-Cell_cycle

Author:
Boris Steipe ( <boris.steipe@utoronto.ca> )
Created:
2017-08-05
Last modified:
2022-09-14
Version:
1.0
Version History:
–  1.0 First live version
–  0.1 First stub
Tagged with:
–  Unit
–  Live
–  Has lecture slides

 

[END]