Gene Control in Eukaryotes

1. Gene Control in Eukaryotes In eukaryotic cells, the ability to express biologically active proteins comes under regulation at several points: 1.Chromatin Structure: The physical structure of the DNA, as it exists compacted into chromatin, can affect the ability of transcriptional regulatory proteins (termed transcription factors) and RNA polymerases to find access to specific genes and to activate transcription from them. The presence modifications of the histones and of CpGmethylation most affect accessibility of the chromatin to RNA polymerases and transcription factors. 2.Epigenetic Control: Epigenesis refers to changes in the pattern of gene expression that are not due to changes in the nucleotide composition of the genome. Literally "epi" means "on" thus, epigenetics means "on" the gene as opposed to "by" the gene.

2. 3.Transcriptional Initiation: This is the most important mode for control of eukaryotic gene expression. Specific factors that exert control include the strength of promoter elements within the DNA sequences of a given gene, the presence or absence of enhancer sequences (which enhance the activity of RNA polymerase at a given promoter by binding specific transcription factors), and the interaction between multiple activator proteins and inhibitor proteins. 4. Transcript Processing and Modification: Eukaryotic mRNAs must be capped and polyadenylated, and the introns must be accurately removed (see RNA Synthesis Page). Several genes have been identified that undergo tissue-specific patterns of alternative splicing, which generate biologically different proteins from the same gene.

3. 5. RNA Transport: A fully processed mRNA must leave the nucleus in order to be translated into protein. 6. Transcript Stability: Unlike prokaryotic mRNAs, whose half-lives are all in the range of 1 to 5 minutes, eukaryotic mRNAs can vary greatly in their stability. Certain unstable transcripts have sequences (predominately, but not exclusively, in the 3'-non-translated regions) that are signals for rapid degradation. 7.Translational Initiation: Since many mRNAs have multiple methioninecodons, the ability of ribosomes to recognize and initiate synthesis from the correct AUG codon can affect the expression of a gene product. Several examples have emerged demonstrating that some eukaryotic proteins initiate at non-AUG codons. This phenomenon has been known to occur in E. coli for quite some time, but only recently has it been observed in eukaryotic mRNAs.

4. 8.Small RNAs and Control of Transcript Levels: Within the past several years a new model of gene regulation has emerged that involves control exerted by small non-coding RNAs. This small RNA-mediated control can be exerted either at the level of the translatability of the mRNA, the stability of the mRNA or via changes in chromatin structure. 9.Post-Translational Modification: Common modifications include glycosylation, acetylation, fatty acylation, disulfide bond formations, etc. 10. Protein Transport: In order for proteins to be biologically active following translation and processing, they must be transported to their site of action. 11. Control of Protein Stability: Many proteins are rapidly degraded, whereas others are highly stable. Specific amino acid sequences in some proteins have been shown to bring about rapid degradation.

5. Gene Control in Prokaryotes • genes are clustered into operons: gene clusters that encode the proteins necessary to perform coordinated function • prokaryotic genes that encode the proteins necessary to perform coordinated function are clustered into operons.

6. The lac operon consists of one regulatory gene (the i gene) and three structural genes (z, y, and a). The i gene codes for the repressor of the lac operon. The z gene codes for β-galactosidase (β-gal), for the hydrolysis of the disaccharide, lactose into its monomeric units, galactose and glucose. y gene codes for permease, increases permeability of the cell to β-galactosides. The a gene encodes a transacetylase. • During normal growth on a glucose-based medium, the lac repressor is bound to the operator region of the lac operon, preventing transcription. However, in the presence of an inducer of the lac operon, the repressor protein binds the inducer and is rendered incapable of interacting with the operator region of the operon. RNA polymerase is thus able to bind at the promoter region, and transcription of the operon ensues.

7. The lac operon is repressed, even in the presence of lactose, if glucose is also present. This repression is maintained until the glucose supply is exhausted. The repression of the lac operon under these conditions is termed catabolite repression and is a result of the low levels of cAMP that result from an adequate glucose supply.

8. Transcription • DNA is transcribed to make RNA (mRNA, tRNA, and rRNA) • Transcription begins when RNA polymerase binds to the promoter sequence • Transcription proceeds in the 5' 3'direction • Transcription stops when it reaches theterminator sequence

9. The Process of Transcription Figure 8.7

10. The Process of Transcription Figure 8.7

11. RNA Processing in Eukaryotes Figure 8.11

12. Translation • mRNA is translated in codons (three nucleotides) • Translation of mRNA begins at the start codon: AUG • Translation ends at nonsense codons: UAA, UAG, UGA Figure 8.2

13. The Genetic Code • 64 sense codons on mRNA encode the 20 amino acids • The genetic code is degenerate • tRNA carries the complementary anticodon Figure 8.2

14. The Process of Translation • Components needed to begin translations come together. Figure 8.9

15. The Process of Translation • On the assembled ribosome, at tRNA carrying the first amino acid in paired with the start codon on the mRNA.the place where this firsts tRNA sits is called the p site.A tRNA carrying the second amino acid approaches. Figure 8.9

16. The Process of Translation • The second codon of the mRNA pairs with a tRNA carrying the second amino acids joins to the seconds by a peptide bond. This attaches the polypeptide to the tRNA in the p site. Figure 8.9

17. The Process of Translation The ribosome moves along the mRNA until the second tRNA is in the p site. The next codon to be translated is brought into the a site. The firsts tRNA now occupies the e site. Figure 8.9

18. The Process of Translation • The second amino acid is paired with the start codon on the mRNA. Is release from the e site. Figure 8.9

19. The Process of Translation • The ribosome continues to move along the mRNA and new amino acids are added to the polypeptide Figure 8.9

20. The Process of Translation • When the ribosome reaches a stop codon, the polypeptide is released. Figure 8.9

21. The Process of Translation • Finally, the last tRNA is released ,and the ribosome comes apart. The released polypeptide forms a new protein. Figure 8.9

22. Regulation • Constitutive genes are expressed at a fixed rate • Other genes are expressed only as needed • Repressible genes • Inducible genes • Catabolite repression

23. Operon • Structure of the operon. The operon consists of the promoter (p) and operator (o) sites and structural genes that code for that code for the protein. The operon is regulated by the product of the regulatory gene Figure 8.12

24. Inducible operon (lacoperon) • Receptor active, operon off. The receptors protein binds with the operators, preventing transcription from the operon. Figure 8.12

25. Inducible operon (lacoperon) • Receptors inactive, operon on. When the inducers allolactose binds to the receptors, it can no longer block transcription. The structural genes are transcribed, ultimately resulting the production of the enzymes needed for lactose catabolism. Figure 8.12

26. Repressible operon (trpoperon) • Recept0r inactive, operon on. The repressor is inactive, and transcription and translation proceed, leading to the synthesis of tryptophan. Figure 8.13

27. Repressible operon (trpoperon) • Receptors active, operon off. When the corepressor tryptophan binds to the receptors protein, the receptors binds with the operators, preventing transcription from the operon Figure 8.13

28. Thetrp operon encodes the genes for the synthesis of tryptophan. This cluster of genes regulated by a repressor that binds to the operator sequences. The activity of the trp repressor for binding the operator region is enhanced when it binds tryptophan known as a corepressor. Since the activity of the trp repressor is enhanced in the presence of tryptophan, the rate of expression of the trp operon is graded in response to the level of tryptophan in the cell. • Expression of the trp operon is also regulated by attenuation.

31. Attenuation • The attenuator plays an important regulatory role in prokaryotic cells because of the absence of the nucleus in prokaryoticorganisms. The attenuator refers to a specific regulatory sequence that, when transcribed into RNA, forms hairpin structures to stop transcription when certain conditions are not met

33. CATABOLITE REPRESSION • Many inducible operons are not only controlled by their respective inducers and regulatory genes, but they are also controlled by the level of glucose in the environment. The ability of glucose to control the expression of a number of different inducible operons is called CATABOLITE REPRESSION.

34. (a) Growth on glucose or lactose alone (b) Growth on glucose and lactose combined Catabolite Repression Figure 8.14

35. Lactose + glucose present • Lactose present, no glucose Figure 8.15