1. BIO 1140 – SLIDE #1 Unit 2 – Information flow Unit 2 – What explains the variety of systems and their regulation? DNA RNA Protein The Central Dogma Replication Transcription Translation q Reading n Chapter 13.3,13.4 n Chapter 14 n Chapter 15 q Objectives n DNA Replication in eucaryotes n What is a gene? n Transcription in eucaryotes n Translation in eucaryotes n Regulation

2. BIO 1140 – SLIDE #2 BIO1140 CELL BIOLOGY Examples of netiquette rules concerning peer respect (from Centre for University Teaching) Be sure your device sound is set to “off” at the beginning of class Stay on task. Activities such as Web surfing or gaming may distract classmates. Listen to your classmates if they complain to you that your use is distracting

3. BIO 1140 – SLIDE #3 Unit 2 – Information flow Gene Regulation WHY IS UNDERSTANDING REGULATION THE KEY TO UNDERSTANDING SO MUCH OF LIFE?  Changes in environment or changes in nutrients  Changes in make up of cell is response to signals  In a multicellular organism, cellular differentiation  Conserve energy Gene regulation refers to the regulation of activity and may occur at any level. While the main control is at the level of transcription additional controls are at the posttranscriptional, translational and posttranslational levels.

4. BIO 1140 – SLIDE #4 Regulation of Gene Expression in Prokaryotes Start with a simple, but powerful model. Prokaryotic gene expression reflects life history. In many cases rapid, reversible responses to the environment are observed. q Typically RNA polymerase binds to a DNA sequence 5’ to the gene called the promoter. Within the promoter may be the consensus sequence 5'-TATAAT-3' called a TATA box. q Repressor proteins binding to other regulatory DNA sequences may prevent the gene from being expressed. q Activator proteins binding to other regulatory DNA sequences may turn on expression of the gene. Repressors and activators may regulate the same gene. Unit 2 – Information flow

5. BIO 1140 – SLIDE #5 Unit 2 – Information flow q In prokaryotes many genes are organized into clusters (transcription units) that are implicated in a single function. q At a smaller scale many genes are organized into operons (one or more operons may be found within a transcription unit ) q The operon itself can be considered as a unit of transcription with several genes controlled by a single promoter. In effect an operon is a cluster of genes and DNA sequences involved in their regulation. RNA polymerase binds at the promoter and transcribes all the genes in the operon into one mRNA (called polycistronic because it contains several cistrons--an older definition of a gene used in genetics). q There are an estimated 630 to 700 operons in E. coli. Classic examples are the lac operon for lactose metabolism (3 genes) and the trp operon genes for tryptophan biosynthesis (5 genes). Regulation of Gene Expression in Prokaryotes

6. BIO 1140 – SLIDE #6 lac Operon for Lactose Metabolism q Lactose metabolism in E. coli requires three genes lacZ, lacY and lacA n lac operon contains all three genes and regulatory sequences q lac operon operator sequence is between promoter and lacZ. The operator sequence is the site of repressor binding. q Study of the lac operon allows us to understand the interaction of one metabolic patheway in the context of both positive and negative regulation. It’s a classic system-Jacob and Monod won a Nobel Prize in 1965 for its study! Unit 2 – Information flow

7. BIO 1140 – SLIDE #7 E. coli lac Operon (see notes) Fig. 15-2, p. 323 Unit 2 – Information flow

8. BIO 1140 – SLIDE #8 lac Operon for Lactose Metabolism q Lac repressor stops lac operon expression Encoded by lac I, synthesized in active form n Binds operator, prevents transcription q Allolactose made from lactose when it enters cell, lasts as long as lactose is available n Inducer of lac operon by binding to Lac repressor n Inducible operon because inducer increases expression n We can experimentally mimic this by adding IPTG, an analogue of lactose that is not metabolized, and use a substrate such as X-gal to measure enzyme activity (X it turns blue!!!) Unit 2 – Information flow

9. BIO 1140 – SLIDE #9 Unit 2 – Information flow Lac Operon: For reference only!! (because some people like structures) X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside)

10. BIO 1140 – SLIDE #10 Regulation of Inducible lac Operon Fig. 15-3, p. 324 Unit 2 – Information flow

11. BIO 1140 – SLIDE #11 Positive Regulation of lac Operon q lac operon operates when lactose but not glucose is present n Glucose more efficient energy source than lactose q Catabolite Activator Protein (CAP) is an activator that stimulates gene expression n CAP activated by cAMP n cAMP only abundant when glucose levels are low Unit 2 – Information flow q In E. coli many genes/operons have CAP regulation. Why is this important for E.coli ??

12. BIO 1140 – SLIDE #12 Positive Regulation of lac Operon 1 Fig. 15-5, p. 326 Unit 2 – Information flow

13. BIO 1140 – SLIDE #13 Regulation of the lac Operon and other operons in E. coli: a thought experiment Fig. 15-5, p. 326 Unit 2 – Information flow E. coli uses a general sensing system to positively regulate many possible genes or operons. Which one it uses depends on what the cell senses is available. In a complex environment with many possible energy sources how does E. coli choose? Imagine the switch from lactose to.... In the presence of lactose and absence of glucose, cAMP/CAP binds to the lac promoter and facilitates the transcription of the lac operon. We can imagine that of many substrates some will cause de-regulation of catabolic genes as with the lac operon. Then cAMP/CAP will bind to its promoter and facilitate transcription of the appropriate catabolic genes.

14. BIO 1140 – SLIDE #14 Regulation of Transcription in Eukaryotes q In eukaryotes, regulation of gene expression occurs at several levels; n Transcriptional, posttranscriptional, translational, posttranslational q Chromatin structure plays an important role in whether a gene is active or inactive (Unit 1) q Regulation of transcription initiation involves the effects of proteins binding to a gene’s promoter and regulatory sites q Methylation of DNA can control gene transcription (See notes) Unit 2 – Information flow

15. BIO 1140 – SLIDE #15 Regulation of Gene Expression in Eukaryotes Occurs at Many Levels Fig. 15-6, p. 328 Unit 2 – Information flow

16. BIO 1140 – SLIDE #16 Organization of a “Eukaryotic Gene” Fig. 15-8, p. 329 Unit 2 – Information flow q Eukaryotic gene organization allows regulation q Promoter includes TATA box that binds transcription factors q Promoter proximal region upstream of promoter increases transcription q Enhancer further determine maximum transcription rate ( what are Silencers?)

17. BIO 1140 – SLIDE #17 Transcription Complex on the Promoter Fig. 15-9, p. 330 Unit 2 – Information flow How do these factors assemble to initiate transcription?  Bind to TATA box area and recruit RNA polymerase II  Transcriptional initiation complex, low rate (“basal”)  Activators bind to promoter proximal elements and increase transcription rate

18. BIO 1140 – SLIDE #18 Transcriptional Complex on the Promoter Fig. 15-9, p. 330 Unit 2 – Information flow This linear representation is useful, but does it give us a complete view? No because...

19. BIO 1140 – SLIDE #19 Transcription Initiation Regulation q Coactivators bridge enhancer and promoter n Interactions between coactivator, proteins at promoter, and RNA polymerase increase transcription q Repressors oppose effect of activators n Transcription rate depends on activation and repression signals n In a multicellular organism these signals may be external (heat, light etc.) or internal (hormones etc.) n May bind to sites on an activator or coactivator or increase association with histones Overall regulation is a balance of many factors! Unit 2 – Information flow

20. BIO 1140 – SLIDE #20 Combinatorial Gene Regulation: how it may work Fig. 15-11, p. 332 Unit 2 – Information flow

21. BIO 1140 – SLIDE #21 Steroid Hormone Regulation Fig. 15-12, p. 333 Unit 2 – Information flow

22. BIO 1140 – SLIDE #22 Methylation of DNA q DNA methylation adds –CH 3 to cytosine n Gene silencing occurs when DNA methylation is located in promoters n Example: Barr bodies q Genomic imprinting n Permanent silencing of a maternal or paternal allele n Inherited methylated allele is silenced n Methylation maintained as DNA is replicated Unit 2 – Information flow

23. BIO 1140 – SLIDE #23 Posttranscriptional, Translational, and Posttranslational Regulation q Posttranscriptional regulation controls mRNA availability q Translational regulation controls the rate of protein synthesis q Posttranslational regulation controls the availability of functional proteins Unit 2 – Information flow

24. BIO 1140 – SLIDE #24 Posttranscriptional Regulation q Posttranscriptional regulation controls availability of mRNA to ribosomes q Pre-mRNA processing changes which proteins are made n Alternative splicing of introns and exons q mRNA breakdown rates are variable n Mechanism: 5’ UTR or 3’ UTR nucleotide sequences Unit 2 – Information flow

25. BIO 1140 – SLIDE #25 Posttranscriptional Regulation q Masking proteins bind to mRNA to prevent translation n Signal for mRNA activation removes masking proteins during development q Micro-RNA (miRNA) regulates gene expression through RNA interferance (RNAi) n miRNA binds to any complementary mRNA sequence and silences it q Small interfering RNA (siRNA) is from RNA encoded outside the cell’s genome n Often used by viruses Unit 2 – Information flow

26. BIO 1140 – SLIDE #26 RNA Interference (mechanism) Fig. 15-13, p. 336 Unit 2 – Information flow The key: the formation of dsRNA and its effect. This is what you need to know!!

27. BIO 1140 – SLIDE #27 Translational and Posttranslational Regulation q Translational regulation controls rate at which mRNAs are used in protein synthesis n Increasing length of poly(A) tail increases translation of mRNA q Posttranslational regulation controls functional proteins n Chemical modification alters activity of protein n Processes inactive precursers to active proteins n Rate of degradation, ubiquitin pathway. Unit 2 – Information flow

28. BIO 1140 – SLIDE #28 Gene Regulation Summary (and answers?) 1.What is a gene? 2.What is a transcript? 3.How does transcription work? 4.How does a cell regulation transcription? 5.How does regulation respond to external factors? Unit 2 – Information flow