I. Prokaryotic Gene Regulation Figure 1: Operon Operon: a) Regulatory Elements consist of an Operator that serves as the on-off switch for the genes of the operon. Also contains a promoter for the Structural Genes & a promoter for a gene that codes for a regulatory protein. b) Coding Sequence is found downstream from the regulatory elements & contains the structural genes whose transcription is controlled by the activation or inhibition of the operator. Figure 2: lac Operon lac Operon Elements Coding Sequence Promoter: Plac Regulatory Gene Promotor: Pi Structural Genes: lac Z (beta galactosidase cleaves lactose into glucose & galactose), lac Y (galactose permease - aids in transport of lactose across cell membrane.), & lac A (galactose transacetylase). lac operon: 1
Environmental Conditions Affecting lac Operon Lactose Absent, Glucose Present: mrna is transcribed from the regulatory gene which produces a Lactose Repressor protein that binds to the operator (activating the off switch ), preventing transcription of the coding sequence. Figure 2.1: Inhibition of lac Operon Lactose present, Glucose Present: some lactose molecules enter the cell & are converted to allolactose by the few molecules of beta galactosidase. Allolactose binds to an allosteric site on the lactose repressor, changing its shape. This causes the repressor protein to dissociate from the operator & set the stage for the transcription of the coding sequence. Figure 2.2: Removal of Repressor Protein 2
Lactose present, Glucose ABSENT: transcription of the structural genes can finally commence due to the appearance of camp, normally found at low concentrations when glucose is present. camp allosterically activates a protein called CRP, then binds to the CRP binding site between the Plac promoter & the regulatory gene. This binding of active CRP bends the DNA s double helix, strengthening the affinity of the promoter region for RNA polymerase so that the rate of transcriptional initiation accelerates in the presence of lactose. Thus the lac operon is fully active only if lactose is available & glucose levels are low. Figure 2.3: CRP Activation & Transcription of Structural Genes tryp operon: 3
Figure 3: tryp Operon tryp Operon Elements Coding Sequence Promoter: Ptrp Regulatory Gene Promotor: trp R Structural Genes: trp E, trp D, trp C, trp B, trp A (code for production of tryptophan) Environmental Conditions Affecting tryp Operon Tryptophan Absent: mrna is transcribed from the regulatory gene that produces an inactive repressor protein that has no affinity for the operator. During this time, the coding sequence is transcribed & the enzyme Tryptophan Synthase is produced, leading to tryptophan synthesis. Tryptophan Present: at a critical concentration, tryp acts to allosterically binds to the inactive repressor protein, changing its shape & allowing it to bind to the operator. This serves to prevent further the transcription of the coding sequence & discontinue tryptophan synthesis. 4
I. Transposable Elements The majority of noncoding or junk DNA is comprised of sequences capable of replicating themselves & reinserting at other loci within the genome. Such elements are called Transposons / Transposable Elements / or Jumping Genes. Figure 1: Breakdown of Eukaryotic Genome Transposable elements were first described in maize by geneticist Barbara McClintok who observed that transposons could insert into active genes, such as those for kernel color. In doing so, these transposons affected the expression of these genes & the resulting phenotypes. Figure 2: Effects of Transposons on Active Genes in Maize McClintok s research revealed that the insertion of the transposable element (Ds) into a specific locus would cause the chromosome to break, whereby the gene on the fragment were lost & thus unexpressed. Consequently, the kernel cells in which the insertion occurred produced regions of the kernel that were colorless, shrunken, & dull. Figure 2a: Effects of Transposons on Active Genes in Maize 5
If the Ds insertion occurred within the locus for purple kernel color, the allele would be inactivated & the cells would be colorless. If, at any time, the Ds element removed itself from this locus, the allele would be expressed, producing purple cells. Due to the random insertion & removal of Ds at this locus during cell development, a spotted kernel would result. II. Insertion of Transposable Elements Figure 3: Simple Transposons: Cut & Paste Method Simple Transposons are removed from a locus by the enzyme Transposase, whereby they can be inserted into a new location within the genome (even within an active gene). Figure 3a: Replicative Transposons: Copy & Paste Method Replicative Transposons are copied & inserted into an additional locus by the enzymes Transposase & Resolvase. These entities increase in number over time within the gene pool of populations. Figure 3b: Retrotransposons: Reverse Transcription 6
Genes for reverse transcriptase throughout the genome make it possible for the reverse transcription of retrotransposons to DNA. III. Retrotransposons Figure 4: Retrotransposons: SINES & LINES Alu s are SINES that are specific to primates & exist at 1 million copies within the human genome (10% of the genome). It is estimated that one new Alu insertion occurs w/in the genome per 200 human births. 7
Figure 4a: Origin of Alu Alu s evolved from the RNA from the 7SL RNA gene that produces the Signal Recognition Particle. This molecule escorts polypeptides destined for the cell membrane to the ER for processing. Impact of Alu w/in Human Genome IMPACT 1: by inserting into introns, Alu elements may become exons (exonization) & introduce new splicing sites that could lead to the formation of new mrnas & gene products (5% of all alternatively spliced exons are derived from Alu elements). Figure 5: Alu Exonization Some of the new proteins resulting from Alu insertions may not be essential for survival but may prove useful if environmental conditions change thus play an integral role in primate evolution. 8
IMPACT 2: by inserting in active genes, Alu may trigger disease (about.4% of Alu insertions disease). For example, Alu insertion w/in the NF1 locus of chromosome 17 can lead to a condition known as neurofibromatosis, resulting in growths within the nervous tissue, skin, & bones. IMPACT 3: the presence of Alu sequences within the genome can be used to clarify the evolutionary relationships between primate groups sequences unique to a particular group must have occurred after it diverged from a common ancestor & vice versa. Figure 6: Alu & Primate Evolution Alu insertions appeared in the common ancestor to all primates approximately 65 million years ago. The number of shared Alu insertions is a direct measure of the degree of relatedness between primate groups. 9