Bio 402/502 Section II, Lecture 2

1. Bio 402/502Section II, Lecture 2 Nuclear Processes: DNA replication and transcription Dr. Michael C. Yu

2. General concepts of DNA replication Purpose of DNA replication? • Duplicate a cell’s genetic material - with accuracy - why? • How does a cell accomplish this? • Via a complex set of cellular machineries - how do you identify these proteins? • Rate: as high as 1KB per second - difference eukaryotic cells have different genome size • Biological significance of DNA replication? Cells die if they can’t replicate their DNA properly

3. Prokaryotic vs. eukaryotic DNA replication Prokaryotes: • Single origin of DNA replication (circular chromosome) Eukaryotes: • Multiple origins of DNA replication • Occurs during S phase of the cell cycle

4. DNA replication is “semi-conservative” DNA replication: Separation of the two strands Complete replication using each strand as a template for the synthesis of a new “daughter” strand (Figure obtained at www.sparknotes.com)

5. DNA replication is also bi-directional • Replication starts at “origins of replication” or “replication fork” • Can have multiple origins within a chromosome - efficient

6. Identify origins of replication: using yeast as a model organism (Figure obtained from Alberts, 4th ed.)

7. DNA polymerase: the enzyme that makes DNA Prokaryotes (E. coli): • Five DNA polymerases: I, II, III, IV, and V Eukaryotes: • Many more DNA polymerases • Can be broadly categorized into replication or repair Substrate: deoxyribonuceloside triphosphate

8. Common structure of DNA polymerase Enzyme has independent domains Binds DNA as it exits the enzyme Responsible for positioning template correctly at the active site Conserved sequence motif for catalytic active site

9. Understanding the function of domains within DNA polymerase In vivo mutagenesis? • Create mutations within the domain and test its function in vivo In vitro assays? • Biochemical reconstitution of replication assay (bacteria) • Not really able to do so with eukaryotes - too complex.

10. Process of DNA replicatiton 1. Helicase separates both strands of the DNA 5. Ligation of Okazaki fragments by ligase (lagging strand only) 2. Single-stranded proteins bind and maintain separated strands 4. Synthesis of DNA by DNA polymerase 3. Prime with 3’-OH end (difference between leading & lagging strand) (Figure obtained at Ohio State Biosci website)

11. A different view of DNA replication process Things to also consider: chromatin access by trans-factors

12. How do you identify “essential” genes involved in DNA replication? The use of conditional mutants - (e.g. temperature sensitive) Loss of CFU at non-permissive temperature 37°C In vitro DNA synthesis 30°C Mutagenized cells grown on petri dish Good model organisms: yeast and bacteria

13. Transcription by RNA polymerase II: making of messenger RNAs 3’ 5’ 5’ 3’ Only one of the two strands of DNA is transcribed into mRNA

14. Transcription in Prokaryotes • Lacks nucleus: couples transcription with translation

15. Transcription in the eukaryotes • The predominant form of eukaryotic gene expression • Three RNA polymerases in eukaryotes: RNA Pol I: 5.8S, 18S, and 28S rRNA genes RNA Pol II: all protein-encoding genes, snoRNA genes, some snRNA genes RNA Pol III: tRNA genes, 5S rRNA genes, some snRNA and small RNA genes • Major players involved in regulating transcription are transcription factors Several types - basal, activators, co-activators, etc. Cell/tissue specific - achieve cell/tissue specificity

16. Basic Concept of Eukaryotic Gene Expression Activation of gene structure (i.e. chromatin) Transcription initiation Majority of the eukaryotic gene expression controls Transcription elongation Process of transcripts Export of mRNAs to the cytoplasm Translation of mRNAs to proteins

17. Basic Concept of Eukaryotic Gene Expression What is the role of chromatin during transcription? • Euchromatin vs. heterochromatin First step - open up chromatin structure

18. General structure of chromatin resulting in its compactness

19. Subunit of all chromatin: nucelosome A nucleosome is composed of DNA & histones A nucleosome wraps approx. 200bp DNA How do you know if a segement of DNA is protected/wrapped within a nucelosome? Difference in their susceptibility to MN (Lewin, Genes IX)

20. Histone N-terminal tails are post-translationally modified (Lewin, Genes IX) Two copies of each core histones per nucleosome N-terminal tails of histones are positioned outside of a nucleosome

21. Model of chromatin regulation during transcription Histone PTMs: a major control in chromatin structure Modification status indicative of transcriptional state (Li et al, Cell, 2007) Which histone modifications correlates with gene activation or silencing?

22. Initiating transcription at the promoter region is a multi-step process Normally, chromatin is silent Histone modifications and their implications on gene expression: Acetylation- transcriptionally active Methylation (K,R)- transcriptionally silent Phosphorylation (S) - activation Ubiquintination - signals methylation Sumolyation - transcriptionally silent (Lewin, Genes IX)

23. Events leading to transcription initiation at the promoter 1. Recruitment of activator (trans) to the cis-element 2. Activators recruit chromatin remodelers 3. Modification of chromatin, reorganization of nucleosome, release of chromatin compactness (Lewin, Genes IX)

24. Recognition of core promoter elements by transcription factors Selectivity of promoters determines which transcription factors are recruited to activate gene transcription. How would you determine the consensus promoter sequences of a gene? (Thomas & Chiang, 2007)

25. Identify cis-acting elements using mutagenesis approach • Approaches: linker scan, alanine substitution, deletion, etc. a) Linker scan approach b) Assays to determine the effect of mutation (Hou et al, 2002)

26. Recruitment of RNA polymerase II: start of transcription initiation Transcription factors specifies the location of transcription and recruits RNA pol II. How would you determine which factors are responsible for transcription in a cell? (Lodish et al, 2000)

27. Protein-protein interactions between transcription factors Principles of a two-hybrid assay: • Use a known transcription factor as a “bait” Pros & cons: • Let cells do the work • More in vivo context • False positives • Organism specific (lacking PTM)

28. Identifying protein-protein interactions between transcription factors • Detect biochemical association between proteins using Co-IP Use mass spec to identify co-IP’ed proteins • Can also use epitope-tagged proteins such as Myc, HA, FLAG, etc

29. Modes of transcription factor activation How would you determine binding of a TF to a promoter? (Lewin, Genes IX)

30. Detecting DNA-protein interactions in vitro • Can use EMSA (electrophoresis mobility shift assay) or gel-shift assay: or PAGE (Sigma website) An example of EMSA Radio-labeled DNA probes (i.e. specific promoter elements) • Use of EMSA involved biochemically purified components/extracts • How would you use this method to determine which mode of transcriptional activation

31. Detecting DNA-protein interactions in vivo 1. Crosslink Protein-DNA complexes in situ 2. Isolate nuclei and fragment DNA (sonication or digestion) 3. Immunoprecipitate with antibody against target nuclear protein and reverse crosslinks 4. Identify DNA sequence by quantitative or real-time PCR 5. Detection of PCR products by PAGE or real-time machine MCS