BIO 308 - Differential Gene Expression - Dr. Daley

Eukaryotic versus Prokaryotic Genes

• Eukaryotic genes are contained in complexes called chromatin

– DNA and protein

– Nucleosomes - basic unit of chromatin

• Octamer of histone (2- H2A-H2B and 2- H3-H4)

• Histone is wrapped with ≈ 140 base pairs of DNA

– Typically wound into tight solenoids - Histone H1 stabilizes these

• H1 is found in the” linker” DNA (60 bases) between nucleosomes

Nucleosome & Chromatin Structure

Eukaryotic versus Prokaryotic Genes

• Eukaryotic genes are not co-linear with their peptide products

• Single strands of eukaryotic mRNA come from non-contiguous regions of the chromosome

Anatomy of a Gene - b Globin

• Exons - regions of the DNA coding for proteins (exits the nucleus)

• Introns - intervening regions of DNA that have nothing to do with the amino acid sequence of the protein

Anatomy of a Gene - b Globin

• Promoter - region which is responsible for binding of RNA polymerase and for the subsequent initiation of transcription

• Transcription initiation site (cap sequence) - represents the 5’ end of RNA - receives a “cap” of modified nucleotides after being transcribed

• Translation initiation site - ATG - 50 base pairs beyond transcription initiation site (5’ untranslated region or leader sequence)

Anatomy of a Gene - b Globin

• First exon - 90 base pairs codes for AAs 1-30 of b-globin

• Next an intron of 130 base pairs - allows the RNA to be processed into mRNA & exit the nucleus

• Exon 2 - 222 base pairs - codes for AAs 31-104

• Then a large intron 850 base pairs - not part of the globin protein structure

• An exon of 126 base pairs - codes for AAs 105-146

Anatomy of a Gene - b Globin

• Next a translation termination codon(TAA) - ribosome dissociates at this codon & protein released

• A 3’ untranslated region - places a 3” tail on mRNA (poly A tail) -

– 200-300 adenylate residues - Gives stability, allows the RNA to leave nucleus & permits the RNA to translated into protein

b Globin Gene

Promoters and Enhancers

• Promoters - sites where RNA polymerase binds to DNA to initiate transcription

– Usually contain sequence TATA - called TATA box - requires basal transcription factors (helper proteins)

• An enhancer - DNA sequence that can activate the utilization of a promoter - controlling the efficiency & rate of transcription from the particular promoter region

– Function through the binding of specific regulatory proteins called transcription factors (like promoters)

Enhancers and Differential Gene Expression

• 1. Most genes require enhancers for their transcription

• 2. Enhancers are the major determinant of differential transcription in space (cell type) and time

• 3. Enhancers can function far away from the promoter - thus can be multiple signals to determine whether a given gene is transcribed

Enhancers and Differential Gene Expression

• 4. Interaction between proteins at the enhancers site and proteins at the promoter site regulate transcription

• 5. Combinations of transcription factors bring about transcription

• 6.a gene can have several enhancer elements, each turning it on in a different set of cells

• 7. Enhancers can also function at other genes as negative enhancers or “silencers”

Transcription Factors

• Proteins - bind to enhancer or promoter region - interact to activate or repress transcription of a particular gene

– Typically bind to specific DNA sequence

Transcription Factors

• Three domains

– 1. DNA-binding domain - recognizes a particular DNA sequence

– 2. Trans-activating domain - activates of represses the transcription of the gene whose promoter or enhancer it has bound

• Usually this domain enables the transcription factor to interact with proteins involved in binding RNA polymerase

– 3. May have protein-protein interaction domain

• Allows the transcription factor’s activity to be modulated by TAFs (TBP associated factors - TBP is part of the TATA box - promoter) or other transcription factors

Locus Control Regions (LCRs)

• DNA regions that function as “super-enhancers”

• Establish an “open” chromatin configuration - inhibiting the normal repression of transcription over an area spanning several genes

– Mechanism unknown

• Best studied LCR regulates expression of b globin family of genes in humans

– Located upstream of the e member of the b globin family (5’ end)

Methylation and Gene Activity

• The promoters of inactive genes - methylated at certain cytosine residues

– Methylcytosine - stabilizes nucleosomes & prevents transcription factors form binding only in vertebrates ( not in Drosophila & nematodes)

• Good correlation between the presence of methylcytosines in the promoter of a gene and repression of transcription

Methylation of Globin Genes

Translational Regulation

• Some messages are only translated at certain times

– mRNA for the milk protein casein - half-life of 1.1 hours in rat mammary gland

– During lactation - half life increases to 28.5 hours

Translational Regulation

• Oocytes use translational regulation

– Certain messages are set aside during egg development - only used much later after fertilization - like the rate and pattern of early cell divisions

– Activation can be accomplished by removal of inhibitory proteins or by polyadenlation of the message