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Control of Eukaryotic Genes

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1 Control of Eukaryotic Genes
Chapter 19: Control of Eukaryotic Genes

2 The BIG Questions… How are genes turned on & off in eukaryotes?
How do cells with the same genes differentiate to perform completely different, specialized functions? Differential gene expressions 200 different cell types

3 1. DNA packing How do you fit all that DNA into nucleus?
DNA coiling & folding double helix nucleosomes chromatin fiber looped domains chromosome nucleosomes “beads on a string” 1st level of DNA packing histone proteins have high proportion of positively charged amino acids (arginine & lysine) bind tightly to negatively charged DNA from DNA double helix to condensed chromosome

4 Nucleosomes “Beads on a string” 1st level of DNA packing
8 histone molecules Nucleosomes “Beads on a string” 1st level of DNA packing histone proteins 8 protein molecules positively charged amino acids bind tightly to negatively charged DNA Histones leave the DNA transiently during DNA replication and stay with the DNA during transcription. DNA packing movie

5 DNA packing as gene control
Degree of packing of DNA regulates transcription tightly wrapped around histones no transcription genes turned off Heterochromatin (Interphase) darker DNA (H) = tightly packed euchromatin lighter DNA (E) = loosely packed H E

6 Points of control The control of gene expression can occur at any step in the pathway from gene to functional protein 1. packing/unpacking DNA 2. Transcription (most common) 3. mRNA processing 4. mRNA transport 5. translation 6. protein processing 7. protein degradation A typical human cell expresses about 20% of its genes at any given time. About 200 different cell types. Only about 1.5% of our DNA codes for proteins, a small fraction codes for RNA products such as rRNA and tRNA, and the rest is UTRs.

7 (a) Histone tails protrude outward from a nucleosome
Histone Modification Chemical modification of histone tails Can affect the configuration of chromatin and thus gene expression Chromatin changes Transcription RNA processing mRNA degradation Translation Protein processing and degradation DNA double helix Amino acids (N-terminus) available for chemical modification Histone tails Figure 19.4a (a) Histone tails protrude outward from a nucleosome

8 Unacetylated histones
Histone acetylation Acetylation of histones unwinds DNA loosely wrapped around histones enables transcription genes turned on attachment of acetyl groups (–COCH3) to postive charged lysines Neutralized (+) charged tails no longer bind to neighboring nucleosomes transcription factors have easier access to genes The enzymes that acetylate or deacetylate are closely associated with the transcription factors that allow for the binding of the transcription macherinery. (b) Acetylation of histone tails promotes loose chromatin structure that permits transcription Unacetylated histones Acetylated histones

9 DNA methylation Methylation of DNA blocks transcription factors
no transcription  genes turned off attachment of methyl groups (–CH3) to cytosine C = cytosine nearly permanent inactivation of genes ex. inactivated mammalian X chromosome = Barr body Ex. Epigenetic inheritance Inheritance of traits by mechanisms not involving the nucleotide sequence Certain proteins that bind to methylated DNA recruit histone deacytlation enzymes – dual mechanism for turning off genes. IT has been found in mice and plants that deficient methylating enzyme leads to abnormal embryonic development. Methylation patterns are passted on and for every replication of DNA the same genes get methylated

10 Regulation of Transcription Initiation
Chromatin-modifying enzymes provide initial control of gene expression By making a region of DNA either more or less able to bind the transcription machinery

11 2. Transcription initiation
Noncoding control regions on DNA promoter nearby control sequence on DNA binding of RNA polymerase & transcription factors proximal control elements UTR located close to the promoter enhancer distant control sequences on DNA binding of activator proteins “enhanced” rate (high level) of transcription Distal control elements can be thousands of nucleotides upsteam or downstream of a gene or even within an intron.

12 Organization of a Typical Eukaryotic Gene
Enhancer (distal control elements) Proximal control elements DNA Upstream Promoter Exon Intron Poly-A signal sequence Termination region Transcription Downstream Poly-A signal Primary RNA transcript (pre-mRNA) 5 Intron RNA RNA processing: Cap and tail added; introns excised and exons spliced together Coding segment P G mRNA 5 Cap 5 UTR (untranslated region) Start codon Stop 3 UTR tail Chromatin changes RNA processing degradation Translation Protein processing and degradation Cleared 3 end of primary transport Figure 19.5

13 Model for Enhancer action
Enhancer DNA sequences distant control sequences Activator proteins bind to enhancer sequence & stimulates transcription Silencer (repressor) proteins bind to enhancer sequence & block gene transcription Much of molecular biology research is trying to understand this: the regulation of transcription. Silencer proteins are, in essence, blocking the positive effect of activator proteins, preventing high level of transcription. Specific transcription factors – activators or repressors. Turning on Gene movie

14 Transcription complex
Activator Proteins • regulatory proteins bind to DNA at distant enhancer sites • increase the rate of transcription Enhancer Sites regulatory sites on DNA distant from gene Enhancer Activator Activator Activator Coactivator B F E RNA polymerase II A TFIID H Coding region T A T A Core promoter and initiation complex Initiation Complex at Promoter Site binding site of RNA polymerase

15 Combinatorial Control of Gene Activation
Enhancer Promoter Control elements Albumin gene Crystallin Liver cell nucleus Lens cell Available activators expressed gene not Crystallin gene not expressed (a) (b) A particular combination of control elements Will be able to activate transcription only when the appropriate activator proteins are present

16 Coordinately Controlled Genes
Unlike the genes of a prokaryotic operon Coordinately controlled eukaryotic genes each have a promoter and control elements The same regulatory sequences Are common to all the genes of a group, enabling recognition by the same specific transcription factors

17 3. Post-transcriptional control
Alternative RNA splicing Different mRNA molecules produced from the same primary transcript Depends on which RNA segments are treated as introns and exons

18 4. Regulation of mRNA degradation
Life span of mRNA determines amount of protein synthesis mRNA can last from hours to weeks Ex. Long lived hemoglobin & short lived growth factor Determined by sequences towards the 3’ end UTR Enzymatic shortening of poly A tail  removal of 5’ cap  nuclease degrades mRNA RNA processing movie

19 RNA interference Small interfering RNAs (siRNA)
short segments of RNA (21-28 bases) bind to mRNA create sections of double-stranded mRNA “death” tag for mRNA triggers degradation of mRNA cause gene “silencing” post-transcriptional control turns off gene = no protein produced siRNA

20 Blockage of translation
RNA interference by single-stranded microRNAs (miRNAs) Can lead to degradation of an mRNA or block its translation The micro- RNA (miRNA) precursor folds back on itself, held together by hydrogen bonds. 1 2 An enzyme called Dicer moves along the double- stranded RNA, cutting it into shorter segments. One strand of each short double- stranded RNA is degraded; the other strand (miRNA) then associates with a complex of proteins. 3 The bound miRNA can base-pair with any target mRNA that contains the complementary sequence. 4 The miRNA-protein complex prevents gene expression either by degrading the target mRNA or by blocking its translation. 5 5 Chromatin changes Transcription RNA processing mRNA degradation Translation Protein processing and degradation Degradation of mRNA OR Blockage of translation Target mRNA miRNA Protein complex Dicer Hydrogen bond Figure 19.9

21 1990s | 2006 RNA interference “for their discovery of RNA interference — gene silencing by double-stranded RNA” Andrew Fire Stanford Craig Mello U Mass

22 5. Control of translation
Block initiation of translation stage regulatory proteins attach to 5' end of UTR of mRNA prevent attachment of ribosomal subunits & initiator tRNA block translation of mRNA to protein Control of translation movie

23 6-7. Protein processing & degradation
folding, cleaving, adding sugar groups, targeting for transport Protein degradation ubiquitin tagging proteasome degradation The cell limits the lifetimes of normal proteins by selective degradation. Many proteins, such as the cyclins involved in regulating the cell cycle, must be relatively short-lived. Protein processing movie

24 Ubiquitin 1980s | 2004 “Death tag” mark unwanted proteins with a label
76 amino acid polypeptide, ubiquitin labeled proteins are broken down rapidly in "waste disposers" proteasomes Since the molecule was subsequently found in numerous different tissues and organisms – but not in bacteria – it was given the name ubiquitin (from Latin ubique, "everywhere") Aaron Ciechanover Israel Avram Hershko Israel Irwin Rose UC Riverside

25 Proteasome Protein-degrading “machine” cell’s waste disposer
breaks down any proteins into 7-9 amino acid fragments cellular recycling A human cell contains about 30,000 proteasomes: these barrel-formed structures can break down practically all proteins to 7-9-amino-acid-long peptides. The active surface of the proteasome is within the barrel where it is shielded from the rest of the cell. The only way in to the active surface is via the "lock", which recognises polyubiquitinated proteins, denatures them with ATP energy and admits them to the barrel for disassembly once the ubiquitin label has been removed. The peptides formed are released from the other end of the proteasome. Thus the proteasome itself cannot choose proteins; it is chiefly the E3 enzyme that does this by ubiquitin-labelling the right protein for breakdown play Nobel animation

26 Proteasomes Are giant protein complexes that bind protein molecules and degrade them Enzymatic components of the proteasome cut the protein into small peptides, which can be further degraded by other enzymes in the cytosol. 3 The ubiquitin-tagged protein is recognized by a proteasome, which unfolds the protein and sequesters it within a central cavity. 2 Multiple ubiquitin mol- ecules are attached to a protein by enzymes in the cytosol. 1 Chromatin changes Transcription RNA processing mRNA degradation Translation Protein processing and degradation Ubiquitin Protein to be degraded Ubiquinated protein Proteasome and ubiquitin to be recycled Protein fragments (peptides) Protein entering a proteasome Figure 19.10

27 The basis of change at the genomic level is mutation
Duplications, rearrangements, and mutations of DNA contribute to genome evolution The basis of change at the genomic level is mutation Accidents in cell division Can lead to extra copies of all or part of a genome, which may then diverge if one set accumulates sequence changes

28 Duplication and Divergence of DNA Segments
Unequal crossing over during prophase I of meiosis Can result in one chromosome with a deletion and another with a duplication of a particular gene Nonsister chromatids Transposable element Gene Incorrect pairing of two homologues during meiosis Crossover and

29 Evolution of Genes with Related Functions: The Human Globin Genes
The genes encoding the various globin proteins Evolved from one common ancestral globin gene, which duplicated and diverged Ancestral globin gene  2 1  G A  -Globin gene family on chromosome 16  -Globin gene family on chromosome 11 Evolutionary time Duplication of ancestral gene Mutation in both copies Transposition to different chromosomes Further duplications and mutations

30 Evolution of Genes with Related Functions: The Human Globin Genes
Subsequent duplications of these genes and random mutations Gave rise to the present globin genes, all of which code for oxygen-binding proteins Ancestral globin gene  2 1  G A  -Globin gene family on chromosome 16  -Globin gene family on chromosome 11 Evolutionary time Duplication of ancestral gene Mutation in both copies Transposition to different chromosomes Further duplications and mutations

31 Evolution of Genes with Novel Functions
The copies of some duplicated genes Have diverged so much during evolutionary time that the functions of their encoded proteins are now substantially different Ex: similar amino acid sequence in lactalbumin and lysozyme enzyme

32 Rearrangements of Parts of Genes: Exon Duplication and Exon Shuffling
A particular exon within a gene Could be duplicated on one chromosome and deleted from the homologous chromosome

33 Portions of ancestral genes TPA gene as it exists today
In exon shuffling Errors in meiotic recombination lead to the occasional mixing and matching of different exons either within a gene or between two nonallelic genes EGF Epidermal growth factor gene with multiple EGF exons (green) F Fibronectin gene with multiple “finger” exons (orange) Exon shuffling duplication K Plasminogen gene with a “kfingle” exon (blue) Portions of ancestral genes TPA gene as it exists today Figure 19.20

34 How Transposable Elements Contribute to Genome Evolution
Movement of transposable elements or recombination between copies of the same element Occasionally generates new sequence combinations that are beneficial to the organism Some mechanisms Can alter the functions of genes or their patterns of expression and regulation


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