3 A. Base-pair substitutions: Silent mutation: No effect, usually when the change occurs in the 3rd nucleotide of a codon.Missense mutation: The change causes the wrong amino acid to be inserted. Can be Natural mutation if the new amino acid has a similar structure to the previous aa.Nonsense mutation: Change turns the codon into a stop codon. Results in a truncated protein.
4 B. Insertion/deletion: An extra nucleotide gets added or removed, causing a frame-shift.All amino acids after the insertion/deletion site will be altered!!
5 Multiple DNA repair pathways Base excision repair (BER)Nucleotide excision repair (NER)Mismatch repair (MR)DNA strand cross link repairHomologous recombination (HR)Non-homologous end joining (NHEJ)Level of damage
6 Base Excision RepairThere are different DNA glycosylases, for different types of damaged bases.AP endonuclease recognizes sites with a missing base; cleaves sugar-phosphate backbone.Deoxyribose phosphodiesterase removes the sugar-phosphate lacking the base.
11 Gene Expression We can say that Gene “A” is being expressed if: Gene “A” is being transcribed to form mRNAThat mRNA is being translated to form proteinsThose proteins are properly folded and are in state where they can be used by the cellCells are selective regarding the genes they express, their amounts and timing.Gene expression can be regulated at different stages.
12 Regulation of Gene Expression Gene expression can be regulatedDuring transcription (transcriptional control).During translation (translational control).After translation (post-translational control).
13 Examples of gene expression regulation Transcriptional control:Regulatory proteins affect the ability for the RNA polymerase to bind to or transcribe a particular gene.Translational control:Regulatory proteins can affect the rate of translation.Enzymes can affect the stability of the mRNA.Post-translational control:Translated protein may be modified by phosphorylation or other modifications that alter the protein’s activity, folding or stability.
14 Gene Expression Some genes are constitutively expressed: Expressed equally at all times.However, many other genes are regulated and their expression can be either induced (positive regulation) or repressed (negative regulation).Also, gene expression is not just “on” or “off” - it varies along different levels!
15 Positive Control of Transcription Positive control occurs when a regulatory protein (activator) binds to DNA and increases the rate of transcription of downstream genes.
16 Negative Control of Transcription lacI binds in a way that it inhibits transcriptionNegative control occurs when a regulatory protein (repressor) binds to DNA and decreases the rate of transcription of downstream genes.
17 Transcriptional Control: The operon: a prokaryote modelseries ofgenes andtheir sharedregulatoryelementsgeneproductscontributeto a commonprocess
20 The lac operon, a model for a negative prokaryotic transcriptional regulatory mechanism
21 Lactose metabolism in E. coli Bacteria break down lactose into its component monomers, glucose and galactose, using the enzyme -galactosidase.Bacteria can then use the monomers to generate ATP through cellular respiration.
22 Lactose metabolism in E. coli Gene= lacZ---PROTEIN=B-GALACTOSIDASE FUNCTION=BREAK DOWN LACTOSEGene= lacY---GAL PERMEASE. FUNTION= IMPORT LACTOSE INTO CELLLactose is transported (imported) into the cell by galactoside permease and cleaved into its monomers by -galactosidase.
23 Lactose metabolism in E. coli The expression of the -galactosidase and galactoside permease genes is regulated by lactose and glucose:High levels of lactose induce the expression of the genes, while high levels of glucose repress their expression.
24 The lacZ gene codes for: The lacY gene codes for: The lac operon genes were identified by the researchers Monod and Jacob in a genetic screen in E. coli searching for mutants that were specifically defective in lactose metabolism.The lacZ gene codes for:-galactosidase.The lacY gene codes for:Galactoside permease.The lacI gene codes for:A regulatory protein (a repressor) that normally functions to repress lacZ and lacY gene expression when lactose is absent.
25 The lac operon genesThe lacZ, lacY, and lacI genes are in close physical proximity to each other on the bacterial chromosome.
26 Negative Control of lacZ and lacY Gene Expression The lacI gene codes for a repressor that binds to DNA just downstream of the promoter, physically blocking transcription of lacZ and lacY.
27 Negative Control of lacZ and lacY Gene Expression In the presence of lactose:Lactose binds to the repressor.Lactose-repressor complex releases from DNA.RNA polymerase can now transcribe lacZ and lacY.Thus, lactose induces transcription by preventing the repressor from exerting negative control.
30 The trp operon, another model for a negative transcriptional regulatory mechanism
31 The trp operonContains 5 genes coding for proteins required for the synthesis of the amino acid tryptophan. Also contains a promoter and operator.When tryptophan levels in the cell/media are low, the expression levels of the trp operon genes are high.There are trp repressor proteins in the cell, but they are unable to bind to the trp operator.
32 The trp operonWhen tryptophan levels in the cell are high, tryptophan binds to the repressor and activities it.The activated trp repressor can now bind to the trp operator and block transcription of the trp operon genes.
37 Positive control of the ara operon E. coli can also utilize arabinose, a pentose found in plant cell walls, as an energy source.The ara operon includes:3 genes required for arabinose metabolism.A promoter to which RNA polymerase can bind.An initiator sequence to which an activator protein (AraC) can bind and stimulate transcription.
38 Positive control of the ara operon Expression of the ara operon genes is high only when arabinose levels are high:Allows the E. coli to preserve resources: express only the genes that fit the environmental conditions.
39 Positive control of the ara operon When arabinose levels are high arabinose binds to AraC, allowing it to bind to the initiator region of the ara operon. This induces the binding of the RNA polymerase to the promoter region of the ara operon, initiating the transcription of ara operon genes.
40 Positive control of the lac operon When cAMP levels are low, CAP is inactive and does not bind to the CAP site, preventing efficient transcription.
41 Positive control of the lac operon When cAMP levels are high, CAP is activated, inducing its binding to the CAP site and promoting efficient transcription.