1. Eukaryotic Gene Expression Managing the Complexities of Controlling Eukaryotic Genes

2. Prokaryotes vs. Eukaryotes ProkaryotesEukaryotes Closely related genes are clustered together Related genes are located on different chromosomes Largely transcriptional control Significant transcriptional control; other levels of control possible Larger number of larger-sized genes Trans-acting sequence- specific DNA binding proteins Proximal Cis-acting sequences Cis-acting sequences can be located at significant distances

3. Control Points for Gene Expression in Eukaryotes DNA RNA Protein transcription translation Transcriptional Control Translational Control Post-Translational Control Post-Transcriptional Control

4. Levels of Eukaryotic Chromatin Structure First level of chromatin coiling Nucleosome = DNA + histone proteins Variations in chromatin condensation affect gene activity.

5. Transcriptional Regulation: Effects of Chromatin Structure Decompaction of chromatin: Transcription factors unwind nucleosomes in the area where transcription will begin, creating DNAse I hypersensitive sitesTranscription factors unwind nucleosomes in the area where transcription will begin, creating DNAse I hypersensitive sites RNA polymerase unwinds more nucleosomes as transcription proceedsRNA polymerase unwinds more nucleosomes as transcription proceeds

6. Transcriptional Regulation: Effects of Chromatin Structure Acetylation of histone proteins (adding -CH 3 CO) reduces DNA-histone interaction, permitting transcription factors to bind.

7. Transcriptional Regulation through Histone Modifications Histone Acetyltrans- ferases add acetyl groups to histones Remodeling Enzymes create open regions on promoters Transcription is ON Transcription is OFF Histone Deacetylases remove acetyl groups Histone Methyltrans- ferases add methyl groups

8. Sites of Histone Modifications Histones can be methylated (Me), acetylated (Ac) or phosphorylated (P) at multiple positions on their amino termini.

9. Transcriptional Regulation: Effects of Chromatin Structure DNA Methylation DNA Methylation (adding -CH 3 ) can occur on cytosines at CpG groupings near transcription start sites DNA Methylation (adding -CH 3 ) can occur on cytosines at CpG groupings near transcription start sites Inactive genes have methylated cytosinesInactive genes have methylated cytosines Active genes have demethylated cytosinesActive genes have demethylated cytosines Histone methylation is associated with cytosine methylation Histone methylation is associated with cytosine methylation Acetylation of histones is associated with cytosine demethylationAcetylation of histones is associated with cytosine demethylation

10. Transcriptional Regulation: Histone Methylation induces DNA Methylation Methyltransferase Heterochromatin Protein 1 DNA Methytransferase Applying your Knowledge What happens to Transcription after DNA Methylation? Thumbs Up: Transcription Increases Thumbs Down: Transcription Decreases Thumbs Level: Transcription Remains the Same Thumbs Up: Transcription Increases Thumbs Down: Transcription Decreases Thumbs Level: Transcription Remains the Same

11. Transcriptional Regulation: DNA Methylation induces Histone Deacetylation Applying your Knowledge What happens to Transcription after Histone Deacetylation? Thumbs Up: Transcription Increases Thumbs Down: Transcription Decreases Thumbs Level: Transcription Remains the Same Thumbs Up: Transcription Increases Thumbs Down: Transcription Decreases Thumbs Level: Transcription Remains the Same Methyl-CpG binding protein Histone Deacetylase

12. Transcriptional Regulation: DNA Methylation guided by siRNAs siRNAs

13. Transcriptional Regulation: Control of Initiation Transcriptional Activator Proteins assist in the formation or action of the basal transcription apparatusTranscriptional Activator Proteins assist in the formation or action of the basal transcription apparatus

14. Transcriptional Regulation: Control of Initiation Transcriptional Activator Proteins bind to Enhancer sequences that increase transcriptionTranscriptional Activator Proteins bind to Enhancer sequences that increase transcription –Enhancers can influence promoters at distances of 50 kb or greater due to DNA looping mechanism –Insulators control the direction of enhancer action

15. Motifs in the DNA-Binding Domains of Eukaryotic Transcriptional Activators

16. Transcriptional Regulation: Control of Initiation Transcriptional Repressor Proteins have three possible modes of actionTranscriptional Repressor Proteins have three possible modes of action –compete with activators for DNA binding sites –bind to sites near activator site and inhibit activator contact with basal transcription apparatus –interfere with assembly of basal transcription apparatus

17. Applying Your Knowledge What effect does each of the following have on transcription? Methylation of Cytosines in CpG groups near transcription initiation sitesMethylation of Cytosines in CpG groups near transcription initiation sites Binding of an Activator Protein to an Enhancer SequenceBinding of an Activator Protein to an Enhancer Sequence Acetylation of Histones in the nucleosomes covering a gene sequenceAcetylation of Histones in the nucleosomes covering a gene sequence Thumbs Up: Increases Transcription Thumbs Down: Decreases Transcription Thumbs Horizontal: No change

18. Post-Transcriptional Regulation: Alternative RNA Splicing

19. Post-Transcriptional Regulation: RNA Editing Base substitution after transcription =

20. Translational Regulation: RNA Stability Degradation of mRNA can occur from the 5’ or 3’ endDegradation of mRNA can occur from the 5’ or 3’ end Stability of mRNA depends onStability of mRNA depends on –5’ cap –3’ poly-A tail –5’ and 3’ UTRs: serve as binding sites for regulatory factors –Coding region Example: Hormone prolactin increases the longevity of casein mRNA coding for milk protein in lactating mammalsExample: Hormone prolactin increases the longevity of casein mRNA coding for milk protein in lactating mammals

21. Translational Regulation Masking of mRNAsMasking of mRNAs –Many species store mRNAs in the cytoplasm of the egg. These mRNAs are inactive due to masking by proteins. Fertilization of the egg initiates unmasking and translation of these mRNAs. Availability of specific tRNAsAvailability of specific tRNAs –In the embryonic development of a hornworm, an mRNA is present from day 1 but a specific tRNA needed for its translation is not produced until day 6.

22. Translational Regulation: RNA Silencing

23. Post-Translational Modification: Phosphorylation Addition or removal of a phosphate group is a common way to change protein activity.

24. Post-Translational Modification: Peptide cleavage Proteins that have an inactive form after synthesis are activated by removal of a small number of amino acids. Prothrombin Thrombin Thrombin Cleavage Fibrin polymer (blood clot) Fibrinogen Fibrin Cleavage Activation of blood clotting factors by cleavage

25. Applying Your Knowledge Which type of control is demonstrated by Alternative RNA splicing mechanisms that give rise to different protein products?Alternative RNA splicing mechanisms that give rise to different protein products? Addition of a phosphate group to activate a protein?Addition of a phosphate group to activate a protein? Increased stability of mRNA in the presence of a regulator molecule?Increased stability of mRNA in the presence of a regulator molecule? Binding of a repressor protein to the regulatory promoter so that an activator protein is unable to bind to this site?Binding of a repressor protein to the regulatory promoter so that an activator protein is unable to bind to this site? 1.Transcriptional Control 2.Post-Transcriptional Control 3.Translational Control 4.Post-Translational Control