Ch. 16 Gene Regulation in Eukaryotes
Methylation
A methyl group is covalently added to the 5′ carbon of cytosine, forming 5-methylcytosine Catalyzed by DNA methyltransferase Usually occurs in regions rich in C and G doublets, called CpG islands (often found in promoters) Regions are often scattered near promoters. Methylation patterns are heritable. When DNA replicates, a maintenance methylase catalyzes formation of 5-methylcytosine in the new strand. Sometimes the methylation pattern is altered because it is reversible. Demethylase catalyzes removal of methyl groups. These are present so methylation is reversible
Example of Genomic Imprinting
A region on human chromosome 15 called 15q11 Rarely, a chromosome deletion results in the baby having only the male or female version of the gene. Male pattern results in Angelman syndrome, with epilepsy, tremors, and constant smiling Female pattern results in Prader-Willi syndrome, marked by muscle weakness and obesity The gene sequences are the same in both cases; the epigenetic patterns are different.
Regulation
After transcription, eukaryotic gene expression can be regulated in the nucleus before mRNA export, or after mRNA leaves. Control mechanisms include alternative splicing of pre-mRNA, gene silencing, translation repressors, and regulation of protein breakdown.
Gene regulation in eukaryotes
Just like in prokaryotes: Negative regulation: a repressor protein prevents transcription Positive regulation: an activator protein stimulates transcription
One type of heterochromatin is the inactive X chromosome in mammals
Males (XY) and females (XX) contain different numbers of X-linked genes, yet for most genes transcription rates are similar. Early in development, one of the X chromosomes in females is inactivated by jamming in all into heterochromatin. All sex linked genes on that chromosome are inactivated. Which X chromosome gets inactivated is random in each cell. The inactivated X chromosome is heterochromatin, and shows up as a Barr body in human female cells. The DNA is heavily methylated, and unavailable for transcription, except for the Xist gene.
What determines if gene expression will occur?
Many inputs determine if gene expression will occur Often there are many transcription factors involved. The combination of factors present determines the rate of transcription. Although the same genes are present in all cells, the fate of the cell is determined by which of its genes are expressed.
Effects of DNA methylation
Methyl groups in promoter regions attract proteins for transcription repression. Methylated genes are often inactive. In development, early demethylation allows many genes to become active Later, some genes may be "silenced" by methylation Silent genes may be turned back on. DNA methylation can play a role in cancer: oncogenes get activated and promote cell division tumor suppressor genes can be turned off
Additional types of histone modification
Methylation: inactivates genes Phosphorylation: effects depend on which amino acids are involved, can go both ways All the epigenetic effects are reversible, so gene activity may be determined by very complex patterns of histone modification. The pattern you had two years ago may not be the pattern you have today.
MicroRNAs
MicroRNAs(miRNAs): small RNAs produced by noncoding regions of DNA. First found in C. elegans. Two genes effect transition through the larval stages: Mutations in lin-14 caused the worm to skip the 1st stage; normal role is to facilitate stage 1 events.
How do eukaryotes coordinate expression of sets of genes?
Most have their own promoters, and may be far apart in the genome. If the genes have common regulatory sequences, they can be regulated by the same transcription factors. In bacteria things are grouped into operons, defined units all controlled by same elements In Eukaryotes the genes can be distributed through out a chromosome or chromosomes, but still regulated by same kind of regulation factors.
Position of regulatory sequences
Position of regulatory sequences can be near... or far! Most regulatory sequences are located near the transcription start site. Others may be located thousands of base pairs away. Transcription factors may interact with the RNA polymerase comaplex and cause the DNA to bend.
Post-translational regulation: proteolysis
Protein content of a cell is a function of synthesis and degradation. Proteins can be targeted for destruction when ubiquitin is attached to it. The complex binds to a proteasome, a large complex where the ubiquitin is removed and the protein is digested by proteases. This is your fail safe, destroying the protein Ubiquitin tag marks the protein for death then proteasome will chop down protein into amino acids, recognition event depends on presence of ubiquitin tags, no folding of protein (as proteins age or get exposed their structure falls apart loses activity to recycle this non functional protein and reuse amino acids ubiquitin attacks and then proteasome recognizes and degrades it. By controlling ubiquitin step you can control proteins at another level) This system is similar to one in bacteria, mechanism is the same
RNA Polymerase II
RNA pol II transcribes protein-coding genes Things that add tail, cap, and splice are attached to RNA polymerase, get to work really fast!
Transcription initiation in eukaryotes
RNA polymerase II can only bind to the promoter after general transcription factors have assembled at the promoter. General transcription factors seek out promoter thus they must bind first. TFIID binds to TATA box Other factors bind to form an initiation complex.
Small interfering RNAs
Small interfering RNAs (siRNAs) also result in RNA silencing. Often arise from viral infection and transposon sequences (pieces of DNA that can jump around from organism to organism). They bind to target mRNA and cause its degradation. May have evolved as defense to prevent translation of viral and transposon sequences.
Sequence-specific regulation
Some regulatory sequences are common to promoters of many genes, such as the TATA box. Some sequences are specific to a few genes and are recognized by transcription factors found only in certain tissues. Many many many regulatory factors that help turn on and off transcription
Eukaryotic promoters
TATA Box Where DNA begins to denature. Duplex starts to pull apart here. TA base pairs only have 2 hydrogen bonds so less energy than 3 hydrogen bonds in GC. In the promoter region. Promoters also include regulatory sequences recognized by a variety of transcription factors. A lot of enzymes to recruit polymerase, to activate it and to stabilize it. This is NOT the same as a -35 and -10 site found in bacteria!
Why bother with transcriptional regulation?
The earlier the cell can stop protein synthesis, the less energy is wasted. Blocking transcription is more efficient than transcribing the gene, translating the message, and then degrading or inhibiting the protein.
miRNAs in humans
The human genome has about 1,000 miRNA encoding regions. miRNAs can inhibit translation by binding to target mRNAs. Each one is about 22 bases long and has many targets, as binding doesn't have to be perfect.
Timing of gene expression
Timing of gene expression is important for development In development of multicellular organisms, certain proteins must be made at just the right time and place. The expression of eukaryotic genes must be precisely regulated. Regulation can occur at several different points.
DNA Polymerase I
Transcribes most rRNA genes
Coordination of Gene Expression
Transcription factor plops down at many different sites to indicate transcription of many different genes responding to the same thing Same thing for silencer.
TFIID
a protein that binds directly to TATA box once in place it recruits "friends" that help stabilize RNA polymerase II
DNA Polymerase III
transcribes tRNA genes
As transcription occurs read template ...
3' to 5' build 5' to 3'
Types of Regulation/Control
1. Remodeling of Chromatin 2. Transcriptional Control 3. Processing control 4. Transport Control 5. mRNA Stability Control
Chromosomal protein alterations or chromatin remodeling
DNA is packaged with histone proteins into nucleosomes. The DNA is inaccessible to RNA polymerase and transcription factors. The histones have "tails" with positively charged amino acids, which are attracted to negatively charged DNA. Histone acetyltransferases add acetyl groups to the tails which changes their charges, and opens up the nucleosome to activate transcription. The consequence is opening region for transcription.
Translational Inhibition by RNAs
Dicer enzyme that recognizes them by recognizing folding in RNA (double stranded), and chops it up Both tiny noncoding RNAs, both have to work with proteins processed by dicer then bind with a protein to form RISC complex that targets mRNA RISC complex = protein + miRNA or siRNA Also happens in bacteria, different mechanism but same overall process
Alternative splicing
Different mRNAs can be made from the same gene depending where splicing occurs. Introns are spliced out; mature mRNAs have none. Sometimes exons are spliced out too (resulting in different proteins). There are many more human mRNAs than there are coding genes. Alternative Splicing Results in Different Mature mRNAs and Proteins Picture: Three very different protein with very different functions based just on splicing. After different functional groups can be added to make changes
Elements that regulate transcription
Enhancers and Silencers Exact regions of enhancers and silencers doesn't matter so much as long as those regions can come back together (Bend)
The Environment and Epigenetic's
Environmental factors can induce epigenetic changes. Monozygotic (identical) twins have identical genomes, and have been used to study epigenetic effects. In 3-year-old twins, DNA methylation patterns are the same. By age 50, when twins have been living apart in different environments, methylation patterns were quite different.
Epigenetics
Epigenetics is the study of changes in gene expression that occur without changes in the DNA sequence. These changes are reversible, but sometimes stable and heritable. Includes two processes: DNA methylation and chromosomal protein alterations. Chemical signatures determine if promoters are going to fire, these changes are reversible and can be passed on, these chemical things can also change over time.
What are the two kinds of chromatin?
Euchromatin and Heterochromatin
Other mechanisms of translational inhibition
GTP cap on 5′ end of mRNA can be modified; if cap is unmodified mRNA is not translated Repressor proteins can block translation directly
Epigenetic Remodeling of Chromatin for Transcription
Histone deacetylases (HDACs) removes the acetyl groups, which represses transcription. In some cancers, genes that inhibit cell division are excessively deacetylated. Drugs that inhibit histone deacetylase may be useful to treat the cancer.
Translational Repression by riboswitches
If iron is around it will interact with the repressor protein and it changes the shape of the repressor protein allowing translation to occur Lots of examples, some repressor protein binds to some metabolite controlling translation of mRNA
Genomic imprinting
In mammals, eggs and sperm develop different methylation patterns which are import for dictating what will happen to the zygote. For about 200 genes, offspring inherit an inactive (methylated) copy and an active (demethylated) one. A race to see which one will win out, which determine if you are healthy or have some genetic problem.
Euchromatin
diffuse, light-staining; contains DNA that is actively transcribed
Xist gene
RNA transcribed from Xist (X inactivation-specific transcript) binds to the chromosome, spreading the inactivation. This RNA is an example of interference RNA. Cascade effect, the endpoint of this gene is to make a functional RNA that works at the level of gene regulation.
Silencers
bind transcription factors that repress transcription. Decrease rate of transcription.
Heterochromatin
condensed, dark-staining, contains genes not transcribed, not actively transcribed
Transcription factors...
control initiation. For an enhancer to work DNA has to loop back around.
Patterns of DNA methylation may include...
large regions or entire chromosomes
lin-4 and lin-14
lin-4 mutations caused some cells to repeat a development pattern normally shown in the 1st stage; its normal role is negative regulation of lin-14. lin-14 encodes a transcription factor that affects genes involved in larval cell progression. lin-4 encodes a 22-base miRNA that inhibits lin-14 expression post-transcriptionally, by binding to its mRNA. Complimentary sequence in that 22 base pair, some sequence septicity between 4 and 14 that drives the interaction
Enhancers
regulatory sequences that bind transcription factors that activate transcription or increase rate of transcription. Drive transcription or increase the rate at which it happens