Regulation of Gene Expression Chapter 11 Regulation of Gene Expression

11.1 Several Strategies Are Used to Regulate Gene Expression Gene expression is precisely regulated Constitutive genes-are actively expressed all the time Inducible genes-are expressed only when their proteins are needed by the cell We will focus on inducible genes

Genes are subject to positive and negative regulation DNA can be regulated at every step from genes to proteins During transcription there is an important form of gene regulation Genes that get transcribed are said to be active

Differential gene expression: expression of different genes in different kinds of cells, despite having the same genome

Two types of transcription factors (proteins) control whether or not a gene is active Repressors Activators These bind to specific DNA sequences at or near the promoter and decide which genes to be transcribed

In negative regulation, a repressor binds near the promoter to prevent transcription In positive regulation, the binding of an activator stimulates transcription

http://www.sciencedirect.com/science/article/pii/S002555641200003X

11.2 Many prokaryotic genes are regulated in Operons Prokaryotes conserve E by making proteins only when needed The most efficient means of regulating gene expression is at the level of transcription

Regulating gene transcription Inducible genes expression is switched on by an inducer Example: prokaryotic bacteria do not create lactase if allactose (lactose) is not present. When allactose (inducer) is present the bacterial cell begins creating the protein lactase to metabolize the lactose (done with three proteins)

Lac Operon http://missbakersbiologyclass.com/blog/2010/05/03/themes-in-biology-regulation/

Operons are units of transcriptional regulation in prokaryotes Structural genes-a gene that encodes the primary structure of a protein not involved in the regulation of gene expression (example three genes that metabolize lactose) A cluster of genes with a single promoter is called an operon and the operon that codes for the three-lactose metabolizing enzymes is called the lac operon

Lac Operon http://missbakersbiologyclass.com/blog/2010/05/03/themes-in-biology-regulation/

The repressor proteins can bind very tightly with the operator The lac operon had another DNA sequence called an operator, which is near the promoter and controls transcription of the structural genes The repressor proteins can bind very tightly with the operator An inducible operon is turned off unless needed A repressible operon is turned on unless not needed

Lac Operon http://missbakersbiologyclass.com/blog/2010/05/03/themes-in-biology-regulation/

Operator-repressor interactions regulate transcription in the lac and trp operons In inducible lac operons, a repressor protein prevents transcription until the lac-encoded proteins are needed The trp operon is a repressible operon that is turned off by a repressor only under some circumstances

Lac Operon The lac operon is not transcribed unless lactose is the main sugar present A repressor protein is normally bound to the operator, preventing transcription When lactose is present it detaches from the operator which allows RNA polymerase to bind to the promoter and transcribe the structural genes

http://www. google. com/imgres http://www.google.com/imgres?q=operon&hl=en&safe=active&tbo=d&biw=1024&bih=566&tbm=isch&tbnid=_EqT6omOTQGqLM:&imgrefurl=http://www.tumblr.com/tagged/lac-operon&docid=eJT3XNPJ7cWwxM&imgurl=http://media.tumblr.com/tumblr_m6k1ewci511qcih5l.gif&w=500&h=499&ei=Nym9UJvEMMvHqQGwroGoBQ&zoom=1&iact=hc&vpx=658&vpy=99&dur=3108&hovh=224&hovw=225&tx=112&ty=144&sig=101186996142079840199&page=1&tbnh=111&tbnw=111&start=0&ndsp=19&ved=1t:429,r:5,s:0,i:99

The key to this system is the repressor protein The repressor is always present in the cell to occupy the operator and keep the operon turned off Has a sport for the inducer to bind and when it binds allosteric regulation happens

Repressor http://en.wikipedia.org/wiki/Repressor

Lac operon animation

trp Operon A repressible operon is switched off when its repressor is bound to its operator In this case the repressor binds to the DNA only if a co-repressor is present Co-repressor-a molecule that binds to the repressor which causes it to bind to the operator and inhibit transcription

trp operon http://en.wikipedia.org/wiki/Trp_operon

The trp operon’s structural genes catalyze the synthesis of the amino acid tryptophan and is a repressible operon In high concentrations the cell wants to stop making the enzymes to create trp Trp functions as a co-repressor that binds to the repressor of the trp operon and prevent transcription

trp operon http://en.wikipedia.org/wiki/Trp_operon

Summarize the differences between operons Inducible: the inducer interacts with the repressor which causes it to not bind to the operator and halts transcription Repressible: the product of the pathway which is the co-repressor binds to the repressor which then binds to the operator and halts transcription

Lac Operon http://missbakersbiologyclass.com/blog/2010/05/03/themes-in-biology-regulation/

trp operon http://en.wikipedia.org/wiki/Trp_operon

RNA polymerase can be directed to a class of promoters Sigma factors-proteins in prokaryotes that can bind to RNA polymerase and direct the polymerase to specific promoters Example: when nutrients run out for bacteria the cell goes into a sporulation lifestyle and is dormant for a while. These genes are only created when directed by sigma factors

trp Operon

11.3 Eukaryotic Genes Are Regulated by Transcription Factors and DNA Changes

Transcription factors act at eukaryotic promoters A eukaryotic promoter is a region of DNA where RNA polymerase binds The most common promoter is the TATA box because it has a lot of A-T base pairs RNA P cannot bind to the promoter unless it has general transcription factors bind to the promoter first

General Transcription Factors http://www.mun.ca/biology/desmid/brian/BIOL3530/DB_Ch09/DBNDiff.html

First, the protein TFIID binds to the TATA box which changes the shape of the DNA so other TF’s can bind The RNA P then binds after other TF’s have bound to the DNA These TF’s may be positive regulators (activators) or negative regulators (repressors) of transcription

General Transcription Factors http://www.mun.ca/biology/desmid/brian/BIOL3530/DB_Ch09/DBNDiff.html

DNA sequences that binds activators are called enhancers, those that bind repressors are called silencers When these bind to DNA sequences they interact with the RNA P complex and cause the DNA to bend The combination of factors present determines the initiation of transcription (2,000 different TF’s in humans so many different regulation possibilities)

Initiation of transcription Example of Activation

Example in book lets read… (pg 217) The expression of sets of genes can be coordinately regulated by transcription factors Eukaryotes also use sigma factors to guide the RNA P to specific promoters The expression of genes can be coordinated if they share regulatory sequences that bind the same transcription factors Example in book lets read… (pg 217)

Epigenetic changes to DNA and chromatin can regulate transcription Euk- can also regulate the transcription of large stretches of DNA Do this by altering the DNA or the chromosomal proteins that package the DNA (this is reversible unlike mutations) These are called epigenetic changes and can be passed on to daughter cells after mitosis or meiosis

Proteins that package the DNA

DNA methylation Cytosine residues in the DNA are chemically modified by the addition of a methyl group (-CH3) to make 5-methyl cytosine This is done by the enzyme DNA methyltransferase Usually done to C’s that are adjacent to G’s Areas that have a lot of the are called CpG Islands

This change is heritable and reversible The methyl group can be removed by an enzyme named demethylase Methylated DNA binds proteins that repress transcription so the gene may be silenced Sometimes large stretches or whole chromosome can be Methylated Euchromatin is transcribed and heterochromatin is Methylated and not transcribed

DNA methylation: represses transcription by condensing DNA --For example, Barr bodies (inactivated X chromosomes in females) often have methylated DNA

Female bar body example… lets read page 219

Histone Protein Modification Another mechanism for epigenetic gene regulation is the alteration of chromatin structure or chromatin remodeling DNA is packaged as a nucleosome which is a core of positively charged histone proteins that DNA is wound around Nucleosomes make it so RNA P cannot bind

Each histone has a tail of amino acids that sticks out of the structure Enzymes called histone acetyltransferases add acetyl groups to these positively charged amino acids which neutralizes their charges Usually there is a strong attraction between the proteins and DNA which is negatively charged This then loosens the nucleosome and allows for transcription

Histone modifications: acetylation promotes transcription due to neutralization of positive charges so they don’t bind to neighboring nucleosomes (I remember “acetylation= activation”)

Acetyltransferases can activate transcription Another chromatin remodeling protein histone deacetylase removes acetyl groups from histones and represses transcription Histone protein modification video

Epigenetic changes can be induced by the environment Epigenetic changes can be passed on to the next generation if done in a germline cell Epigenetic genomes have been proven to be different by twin studies Stress can be a factor for methylation

11.4 Eukaryotic Gene Expression Can be Regulated after Transcription So far we have only talked about gene regulate before transcription, What about after…

Different mRNA’s can be made from the same gene by alternative splicing Most mRNA in eukaryotes have several introns If an exon were to be spliced out when two intons are cut this would create an new protein This is called alternative splicing and is a deliberate way to create alternative proteins from a single gene

Alternative RNA splicing: allows different RNA molecules (and thus, different proteins) to be produced from same primary transcript (pre-mRNA)

Examples on page 221… Before the human genome was sequenced…scientists believed there were 80,000 to 150,000 protein coding genes They now know there are 24,000 genes 80 percent of this variation are due to alternative splicing Example with humans and chimpanzees

Let’s review the kinds of RNA we’ve encountered so far: mRNA tRNA rRNA snRNP And let’s add: miRNA (micro RNA)(also known as RNAi) (Fire & Mello, Nobel Prize 2006) siRNA (small interfering RNA)

MicroRNAs are important regulators of gene expression Only a fraction of genes code for proteins Biologists used to think that the rest of the genome was not transcribed and they called it “junk” DNA They now know that some of it is transcribed into very small RNA molecules called microRNA (miRNA) These usually code for genes that turn on or off gene expression (example page 222)

mRNA degradation Nova clip RNAi

The microRNA targets a mRNA and inhibits its translation It than degrades the target mRNA This is a gene silencing mechanism

Translation of mRNA can be regulated For most genes mRNA doesn’t necessarily relate to the number of proteins A lot of mRNA doesn’t necessarily mean a lot of protein or vice versa Cells regulate how many proteins get made after transcription in 2 ways: Regulation the translation of mRNA Altering how long proteins persist in the cell

3 ways in which translation can be regulated (examples page 223) 1. Inhibition of translation with miRNAs 2. Modification of the 5’ cap An mRNA that is capped with an unmodified GTP molecule is not translated 3. Translational repressor proteins Block translation by binding to mRNAs and preventing their attachment to the ribosome Known as translational repressors

Protein stability can be regulated Protein content is a result of both protein synthesis and protein degradation Proteins can be targeted for destruction in a chain of events that begins when an enzyme attaches a protein called ubiquitin Other ubiquitins then attach to the primary one forming a polyubiquitin

The polyubiquitin then binds to a protein complex called a proteasome This then unfolds the protein inside of it Example the breakdown of cyclins during the cell cycle This needs to be done at the correct time and it is done by proteasomes

Cyclin-dependent kinase, phosphorylates substrate proteins Maturation-promoting factor (or M-phase PF)