Control of Eukaryotic Genome Chapter 19. Control of Eukaryotic Genome 2005-2006 a

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? 2005-2006

Prokaryote vs. eukaryote genome Prokaryotes – small size of genome circular molecule of naked DNA DNA is available to RNA polymerase control of transcription by regulatory proteins operon system most of DNA codes for protein or RNA no introns: small amount of non-coding DNA regulatory sequences: promoters, operators prokaryotes use operons to regulate gene transcription, however eukaryotes do not. since transcription & translation are fairly simultaneous there is little opportunity to regulate gene expression after transcription, so control of genes in prokaryotes really has to be done by turning transcription on or off. 2005-2006

Prokaryote vs. eukaryote genome Eukaryotes large genome how does all that DNA fit into nucleus? DNA packaged in chromatin fibers regulates access to DNA by RNA polymerase cell specialization Genes turned on and off at different times most of DNA in humans does not code for protein 97% non-coding DNA 2005-2006

Points of control The control of gene expression can happen at any point along the way: unpacking DNA transcription mRNA processing mRNA transport translation protein processing protein degradation 2005-2006 a

Why turn genes on & off? Specialization Development each eukaryotic cell expresses only a small fraction of its genes Development different genes are needed at different points in the life cycle of an organism afterwards need to be turned off permanently Responding to organism’s needs homeostasis Response to environment – must turn genes on & off A prokaryote has most of its genes turned on most of the time. Whereas in a multicellular organism, each cell has most of its genes turned off. A brain cell expresses many different proteins than a muscle cell. 2005-2006 a a

DNA packing How does all that DNA fit into nucleus? DNA coiling & folding double helix nucleosomes chromatin fiber 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 2005-2006

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 many positively charged amino acids arginine & lysine bind tightly to negatively charged DNA 2005-2006

DNA packing tightly packed = no transcription = genes turned off darker DNA (H) = tightly packed lighter DNA (E) = loosely packed 2005-2006

DNA methylation Methylation of DNA winds up chromosome tighter – think of “pill bug” no transcription = genes turned off attachment of methyl groups (–CH3) to cytosine C = cytosine can be permanent inactivation of genes ex. inactivated X chromosome 2005-2006 a a

Histone acetylation Acetylation of histones unwinds DNA loosely packed = transcription = genes turned on attachment of acetyl groups (–COCH3) to histones Changes shape in histone proteins Transcription can proceed when “unwound” 2005-2006

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You are more than your DNA EPIGENETICS You are more than your DNA

University of Utah http://learn.genetics.utah.edu/content/epigenetics/intro/

Transcription initiation Control regions on DNA promoter nearby control sequence on DNA binding of RNA polymerase & transcription factors “base” rate of transcription enhancers distant control sequences on DNA binding of activator proteins “enhanced” rate (high level) of transcription 2005-2006 a

Model for Enhancer action Enhancer DNA sequences distant control sequences Activator proteins bind to enhancer sequence & stimulates transcription Silencer 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. 2005-2006 a Gene movie

https://www.youtube.com/watch?v=ysxtZJUeTCE 2005-2006

Post-transcriptional control Alternative RNA splicing different exons removed = different proteins 2005-2006 a

Regulation of mRNA degradation Life span of mRNA determines pattern of protein synthesis mRNA can last from hours to weeks 2005-2006 RNA processing movie a

RNA interference Small RNAs (sRNA) 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” siRNA 2005-2006 a a

functionally turns gene off One of the Newer biology topics RNA interference Small RNAs mRNA double-stranded RNA sRNA + mRNA mRNA degraded functionally turns gene off 2005-2006 a a

RNA Degradation: HHMI Roy Barker https://www.youtube.com/watch?v=d7nmojex01c (3 mins. Only) RNAi: (gene silencing) https://www.youtube.com/watch?v=cK-OGB1_ELE 2005-2006

Control of translation Block initiation stage regulatory proteins attach to 5’ end of mRNA prevent attachment of ribosomal subunits & initiator tRNA block translation of mRNA to protein 2005-2006 Control of translation movie

Protein processing & degradation folding, splitting, adding sugar groups, targeting for transport Protein degradation ubiquitin tagging proteosome 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. 2005-2006 Protein processing movie

Ubiquitin 1980s | 2004 “Death tag” mark unwanted proteins with a label 76 amino acid polypeptide, ubiquitin labeled proteins are broken down quickly 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 2005-2006 a

Proteosome / Ubiquitin (UPS) https://www.youtube.com/watch?v=hvNJ3yWZQbE (~ 6 min.) 2005-2006

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Structure of the Eukaryotic Genome 2005-2006 a a

How many genes? Genes only ~ 3% of human genome protein-coding sequences 1% of human genome non-protein coding genes 2% of human genome tRNA ribosomal RNAs siRNAs 2005-2006 a a

What about the rest of the DNA? Non-coding DNA sequences regulatory sequences promoters, enhancers terminators Non-Coding DNA introns repetitive DNA centromeres telomeres tandem & interspersed repeats transposons & retrotransposons Alu in humans a 2005-2006 a

Genetic disorders of repeats Fragile X syndrome most common form of inherited mental retardation defect in X chromosome mutation causing many repeats of CGG triplet in promoter region 200+ copies normal = 6-40 CGG repeats FMR1 = Fragile X Mental Retardation gene FMRP = Fragile X Mental Retardation Protein a 2005-2006 a

Fragile X syndrome The more triplet repeats there are on the X chromosome, the more severely affected the individual will be mutation seems to increase severity with each generation mutation seems to increase severity with each generation A summary of existing research conducted by the Centers for Disease Control and Prevention in 2001 estimated that approximately one in 3,500 to 8,900 males is affected by the full mutation of the FMR1 gene, and that one in 1,000 males has the premutation form of the FMR1 gene. This study also estimated that one in 250 to 500 females in the general population has the premutation. Another study6 estimated that one in 4,000 females is affected by the full mutation. 2005-2006

Huntington’s Disease Rare degenerative neurological disease 1st described in 1872 by Dr. Huntington most common in white Europeans 1st symptoms at age 30-50 death comes ~12 years Mutation on chromosome 4 – (dominant) CAG repeats 40-100+ copies normal = 11-30 CAG repeats CAG codes for glutamine amino acid When the HD gene was found, researchers discovered that HD belongs to a newly discovered family of diseases called "triplet repeat" diseases. A gene's DNA bears coding molecules which are translated into specific amino acids, the building blocks of proteins. In the HD gene, the coding molecules cytosine, adenine, and guanine (CAG) - the triplet - repeat in a stretch more than they do in the normal gene. Normally the CAG sequence repeats between 11 and 30 times. People with HD may have CAG repeated between 36 and 125 times. The onset of the disease generally is in the 30s and 40s (with a range of age 2 to 82), but more than 60 repeats of CAG often are associated with an onset of HD before age 20. With the majority of people who have fewer than 60 repeats, there is great variability. A person can have 40 CAG repeats in his or her DNA sequence and have onset at age 17 or 70. On average, however, a feature dubbed "genetic anticipation" occurs. Each time the unstable DNA is passed on to offspring, the affected person experiences an earlier onset. 2005-2006 a a

Huntington’s disease Abnormal (Huntington) protein produced chain of charged glutamines in protein bonds tightly to brain protein, HAP-1 Woody Guthrie 2005-2006

Interspersed repetitive DNA Repetitive DNA is spread throughout genome repetitive DNA makes up ~ 25-40% of genome of mammals in humans, at least 5% of genome is made of a family of sequences called, Alu elements 300 bases long Alu is an example of a "jumping gene" – a transposon DNA sequence that "reproduces" by copying itself & inserting into new chromosome locations 5% of genome = millions of copies of this sequence! Alu doesn’t seem to be active anymore. No useful function to organism. Used to study population genetics = relatedness of groups of people a 2005-2006 a

Rearrangements in the genome Transposons piece of DNA that can move from one location to another in cell’s genome One gene of an insertion sequence codes for transposase, which catalyzes the transposon’s movement. The inverted repeats, about 20 to 40 nucleotide pairs long, are backward, upside-down versions of each other. In transposition, transposase molecules bind to the inverted repeats & catalyze the cutting & resealing of DNA required for insertion of the transposon at a target site. 2005-2006

Transposons Insertion of transposon sequence in new position in genome This can cause mutations when they land within the coding sequence of a gene 2005-2006 a

Transposons 1947|1983 Barbara McClintock discovered 1st transposons in Maize = (corn) in 1947 She found that transposons were responsible for a variety of types of gene mutations, usually insertions deletions and translocations 2005-2006 a

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Retrotransposons Transposons actually make up over 50% of the corn (maize) genome & 10% of the human genome. 2005-2006 a a

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Any Questions?? 2005-2006 a a