notes 19

Molecular Biology Chapter 19 Controlling gene expression Like prokaryotes, need to alter expression due to environmental variation But, multicellularity leads to new challenges Different cell types Different tissue types Communication between cells Points of control In the nucleus Chromatin structure At transcription During RNA processing Outside the nucleus (cytoplasm) Rate of mRNA degradation Rate of translation Rate of post-translational modification Chromatin remodeling DNA is highly compacted Chromatin Compacting DNA Histones Proteins with (+) charge DNA has a (-) charge 8 histones/ nucleosome DNA wraps around 1.65 times About 146-147 bases “Beads on a string” Shortens DNA about 7 fold How did they discover this? DNase cuts double stranded DNA DNase cuts DNA that’s easy to reach first Separate DNA by size Histones compact into 30- nm fibers Shortens DNA another 7-fold 30-nm fibers attach to nuclear membrane, form radial loops Most compact DNA- chromosome Formed during cell division 10,000x more compact Protein scaffold in the center Chromosome is made of a pair of sister chromatids Levels of structure If the promoter buried in here, how much transcription will there be? why? How can we test this? Scientific method time Question: how does compacting DNA into 30 nm fibers affect gene expression? Hypothesis: densely packed chromatin prevents RNA polymerase from reaching the promoter Test: ??????????? Relaxing chromatin Not all genes are expressed in all cells In blood cells: Lots of ?-globin No ovalbumin Test with DNase If RNA polymerase can reach the DNA, so can the DNase , and vice versa In blood cells Will DNase cut ?- globin gene? Will DNase cut ovalbumin gene? For the ?-globin gene, DNA is chopped up For the ovalbumin, no cutting, chromatin is left intact What type of control are histones providing, positive or negative? Test by “breaking” the histones- identify mutants that don’t produce them Brewer’s yeast With histone cells, genes are constantly expressed at high levels Histones provide negative control Default state is off Altering chromatin Need the ability to turn genes on/off Relax or condense chromatin at will Chemically modify the histones Acetylation (-CH?COOH) Methylation (CH?) Chromatin- remodeling complexes Multi- protein complexes Restructure chromatin Histone acetyl transferases (HATs) Add acetyl groups Adding acetyl groups relaxes the chromatin Adding acetyl groups= positive control Need to turn the gene off? Histone dacetylases (HDACs) Methylation has variable effects Can be on the DNA itself Can be positive or negative control Epigenetics Variation in cell types can be due to variation in histone & DNA modification Epigenetic inheritance During mitosis, acetylation and methylation patterns are also passed on to daughter cells Muscle cells produce muscle cells, skin cells produce skin cells, etc……. Epigenetic tags change due to environmental variation Alter gene expression to fit current conditions Also, non-Mendelian inheritance Daphnia Exposure to predators leads to morphological change that persists for generations Identical genotypes, different phenotypes What environmental variables affect methylation/ epigenetic imprinting? (Dolinoy et al. 2007) Bisphenol A in the maternal diet Can be counteracted Could you have bisphenol A in your diet? Promoters Site where RNA polymerase binds to the DNA to start transcription Can’t be identified until the chromatin is relaxed Similar to the -35 box and the -10 box in prokaryotes 3 highly conserved sequences (every promoter has 2 of the 3) TATA box TATA-binding protein Regulatory sequences Lots of regulatory sequences Promoter-proximal elements Positive control Like the CAP site in prokaryotes Increases transcription when regulatory molecule binds to it Sequences is unique to specific genes (or sets of genes) Regulatory sequences can be far away from their genes Enhancers Discovered in antibody genes Eukaryotic genes usually have introns Large non-coding regions between genes Could there be non-proximal regulatory sequences in these places? 1. Isolate the gene with the proximal promoter and all the introns 2. Use restriction enzymes to remove specific pieces of the intron or flanking sequences 3. Use DNA ligase to reconnect the fragments 4. Insert the gene into living cells 5. Quantify mRNA production In human cell, high mRNA levels (positive control) In mouse cell with no introduced DNA, no mRNA (negative control) In mouse cell with introduced intact DNA, high mRNA levels (positive control) Removing specific introns can lead to no transcription The removed piece may be 1000s of bases from the proximal promoter! Enhancers Enhancers= requlatory sequences far from the promoter Lots of variation in enhancer structure Don’t need to be near the gene 100,000s of bases away Upstream, downstream, or in an intron Work if orientation is flipped Work if moved Most genes have multiple enhancers Regulatory proteins (Transcription factors) bind to enhancers Speed up transcription Positive control Regulatory sequences Silencers= provide negative control Protein(s) bind to them, transcription stops Similar in structure to enhancers, can be found all over What is a gene? DNA that codes for a protein, along with its regulatory sequences Control of gene expression How do these different elements work together in eukaryotes? Variation between cell types is due to differences in histone modification, differences in regulatory transcription factors Regulatory transcription factors Proteins that bind to proximal promoters, enhancers, silencers Specific to particular cell types and genes Basal transcription factors Interact with the promoter, attract RNA polymerase Required, but not specific, provide little control Transcription control Relax the chromatin Adding acetyl groups (HATs) Will vary depending on the cell types, stage of development, environment, etc… Transcription activators bind to enhancers Basal transcription proteins bind to the promoters RNA polymerase is recruited by basal transcription factors Binding is promoted by enhancers and proteins at the proximal-promoter site (or stopped by proteins binding to silencers) RNA polymerase binds, basal transcription complex forms Includes about 60 proteins Post- transcriptional control Eukaryotic mRNA needs to be processed before translation Removing introns 5’ caps and poly-A tails to influence mRNA’s lifespan Proteins can be turned on/off RNA processing snRNPs bind to the intron 5’ end is GU, 3’ is an A Spliceosome is formed Intron is looped on itself, U is connected to the A Exon fragments are ligated together Post-transcriptional control Intron splicing also controls gene expression Tropomyosin: protein found in muscles 14 exons, 13 introns What if during the splicing, some of the exons are removed, too? Alternative splicing mRNA is spliced in different ways to produce different proteins 1 gene can code for more than 1 protein!! Regulatory proteins interact with the spliceosome to control where splicing occurs Alternative splicing= sourse of variation in proteins produced In humans: about 20,000 genes, but about 50,000 proteins Points of control In the nucleus Chromatin structure At transcription During RNA processing Outside the nucleus (cytoplasm) Rate of mRNA degradation Rate of translation Rate of post-translational modification Post translational control Proteins can bind to mRNAs, preventing them from being translated mRNA in egg cells Ribosome can be stopped by chemical modification Phosphate groups Respond to heat-stress or infection Why stop making proteins in these conditions? Eukaryote vs Prokaryote Eukaryote DNA is packed tightly, must be relaxed for promoter to be accessible Default state is “off” Alternative splicing of mRNA 1 gene can be many different proteins Transcriptional control involved dozens of proteins, very complex Fine control To coordinate expression, genes will use the same regulatory transcription factors Genes can be physically distant Prokaryote No histones, little packing, promoters are always available Default state is “on” No splicing 1 gene= 1 protein Transcriptional control much less complex On or off To coordinate expression, genes form an operon Genes are physically close Case studies on epigenetic control Yeasts Human brain Ecological effects Niche width How many different resources can a species use? Generalist vs specialist species Is niche width a function of genetic diversity? Generalist species have more genetic diversity individually Generalist species care really just a collection of different specialist genotypes? Herrera et al. 2011. Jack of all nectars, master of most: DNA methylation and the epigenetic basis of niche width in a flower-living yeast. Molecular Ecology. Does the yeast use a broad range of resources? Individual yeast strains can use all these sources No “specialists” within the species Are there differences in gene expression depending on the food source? How do you detect differences methylation patterns? Methylation in fungi isn’t random Occurs in 5’- CCGG- 3’ sequence Restriction enzymes Restriction enzymes (or restriction endonucleases) Cut DNA, but only in the middle of specific sequences Sticky vs blunt ends Ecological effects Hypothesis: individual yeast cells turn on/off genes related to sugar metabolism depending upon their environment Experiment: raise yeast on different diets, look for differences in methylation patterns Variables: sugar type, sugar concentration, interaction between type and concentration Prediction: DNA cut with Hpall will produce different sized bands on a gel when the DNA is methylated Correlation between methylation and diet type Is the methylation adaptive? Is it changing gene expression beneficial for yeast in high fructose nectar, for example? Experiment: Turn off methylation and see if yeast are negatively impacted 5-azacytidine is a DNA methylation inhibitor Preventing methylation has huge negative impact in high sugar environments Human brain Patient presents with: Nausea Headaches Vision problems Fatigue What could be wrong with them? Stage IV glioblastoma multiform Cancer of astrocytes (supporting cell in nervous tissue) Temozolomide works on some, but not all, patients MGMT: gene for –methylguanine-DNA-methyltransferase (DNA repair) 1. Is it better to have a methylated or unmethylated promoter? 2. How does methylation affect expression of MGMT? 3. Do these patients have a good prognosis? 4. How else can you treat this cancer?