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Each structural gene has its own promoter and is transcribed independently Monocistronic mRNA - each mRNA codes for a single polypeptide Polycistronic mRNA - the mRNA codes for two or more polypeptides are only found in prokaryotes Chromatin structure affects gene expression Transcription requires unwinding of the DNA from the histone proteins
Eukaryotic cells have many more transcriptional activators as well as repressors Again, transcription and translation are physically separated so the regulatory mechanisms in eukaryotic cells must differ from those in bacteria
DNAse I Hypersensitivity
Use a nuclease as a tool to probe chromatin structure Rationale: DNA tightly wound around nucleosomes will be resistant to the nuclease since the DNA is protected by the histone proteins Changes in chromatin that make the DNA less tightly associated with the nucleosomes will be seen as an increased sensitivity to degradation by a nuclease
DNAse I Hypersensitivity
The DNA located ~1,000 bp upstream of the promoter of transcriptionally active genes is hypersensitive to cleavage by DNAse I Indicates that the chromatin has become more relaxed and the DNA is more loosely associated with the nucleosomes Regulatory proteins and RNA polymerase have better access to the gene promoter
Acetyl group
Flowering in Arabidopsis
Arabidopsis is an important genetic model for plant systems Text describes an example of controlling expression of genes involved with flowering that involves acetylation and deactylation and its effect on transcription Read this section for a better understanding of this regulatory process (see Figure 17.3).
DNA Methylation
Methylation of cytosine at position 5 is another important mechanism for affecting transcription Heavily methylated DNA is associated with repression of transcription Unmethylated DNA is associated with DNA that is available for transcription Usually occurs in CpG islands
CpG Islands
Simply means that a methylated C is usually followed by a G The p represents the phosphodiester bond Inactive genes have methylated CpG Find heavy methylation of CpG with fully repressed genes such as those on the inactive X-chromosome of mammalian females
Chromatin Remodeling
Some transcription factors or other regulatory proteins alter chromatin structure without affecting histones directly For example, nucleosomes can be repositioned to allow binding and initiation of transcription
Epigenetics
Changes to DNA and chromatin that affect traits that are passed on to other cells or even future generations But not caused by changes in DNA sequence Example - maternal behavior The offspring of rats where the mother licks and grooms them more show less fearful and stressrelated responses as adults Additional examples in text
Molecular Mechanism
Changes in chromatin must be stable through chromosome replication if the epigenetic effect is inheritable Key mechanism appears to be DNA methylation CpG methylation means there are methylated cytosine on opposite strands After replication, only the old strand is methylated hemimethylated Methyltransferase methylates adds -CH3 tounmethylated new strand siRNA, miRNA and piRNA also play a role in some cases
Transcriptional activators usually bind to either: A regulatory promoter that is upstream of the core promoter An enhancer, which may be located several thousand bases from the promoter
The DNA binding site for GAL4 protein is called UASG for upstream activator sequence for GAL4 Binding of GAL4 to UASG activates transcription GAL4 activity is regulated by GAL80 In the absence of galactose, GAL80 binds to GAL4, which prevents binding to UASG and no transcription
In the presence of galactose, GAL80 binds galactose Allosteric change prevents binding of the GAL80-galactose complex to GAL4 GAL4 is free to bind to the UASG and transcription initiates
No galactose
+ galactose
Insulators
Most enhancers could affect any gene within their proximity Insulators (also called boundary elements) are DNA sequences that function to limit (or isolate) the activity of an enhancer
Response Elements
Each heat shock gene shares a common regulatory DNA sequence called a response element Usually have a short consensus sequence Gene may have additional response elements
One gene can be activated by a variety of stimuli by have several different response elements
mRNA Processing
Alternative splicing of exons of the pre-mRNA can yield different proteins Use an example from Drosophila Sxl (sex lethal) protein is synthesized in female cells Sxl is an RNA binding protein and binds to the tra (transformer) pre-mRNA and affects splicing
In the absence of sxl protein (males), splicing of the tra mRNA occurs at a different place In females, the sxl protein binds to the pre-mRNA to prevent splicing Splicing at an alternative site Result is mRNA in males codes for an inactive tra protein The alternative splicing in females leads to a tra mRNA that codes for a functional protein
RNA Stability
Eukaryotic mRNA is, in general, more long lived than prokaryotic mRNA Some eukaryotic mRNAs are stable for hours, days or even months Can results in very large differences in the amount of protein made Degradation usually begins at the cap structure on the 5 end
RNA Silencing
A recently discovered mechanism for silencing gene expression by targeted degradation of the mRNA Possibly as many as 30% of genes utilize RNA silencing Noble prize awarded in 2006