A wheel of mass M has radius R. It is standing vertically onthe floor, and we want to exert a horizontal force F at itsaxle so that it will climb a step against which it rests 98.(Fig. 10-66). The step has height h, where h < R. Whatminimum force F is needed?FIGURE 10-66 93.
Chapter 14: Gene Regulation - Gene regulation: the level of gene expression can vary under different conditions - Almost all genes are regulated so expression depends on environment - Genes that are unregulated are called constitutive o have essentially constant levels of expression o constitutive genes often encode proteins that are continuously necessary for the survival of the organism - Benefit of gene regulation o you aren’t making products that aren’t needed o Or creating proteins that interact negatively with each other - Gene regulation is important for: o Metabolism o Response to environmental stress o Cell division - Regulation can occur at any point in the pathway of gene expression - Most common way of regulating gene expression in bacteria is by influencing INITIATION of transcription o Initiation: ability of RNA polymerase to bind to promoter or start elongation o Transcription can be increased or decreased o 2 main types of regulatory proteins: § Repressors: Bind to DNA and inhibit transcription • Negative control • An inducer will bind to the repressor causing it to be released from the DNA allowing RNA polymerase to transcribe the gene (conformational change) • Corepressor can bind to repressor, causing a conformational change that causes it to bind to the DNA and inhibit transcription § Activators: Bind to DNA and increase transcription • Positive Control • Needs inducer to bind to DNA (causes conformational change) • Inhibitor binds to activator, causing it to be released from the DNA à increases transcription § Regulatory proteins have two binding sites • One for effector molecule (inducer) • One for the DNA § Corepressors can bind to repressor and allow repressor to bind § Inhibitor can bind to activator and not allow the activator protein to bind - Lac Operon o Operon is a regulatory unit with a few genes under the control of one promoter § Encodes polycistronic mRNA: codes for multiple (two or more) structural genes § Allows for the coordination of a group of genes that have a common functional goal o Operon has a promoter, terminator, structural genes and operator o LacI gene: encodes repressor § Not a part of the lac operon o 3 protein encoding genes: Z, Y, A § Lac Z encodes beta galactosidase • Enzyme that breaks down lactose into allolactose § Lay Y encodes lactose permease • enzyme brings lactose into the cell § Lac A encodes galactoside transacetylase • Enzyme allows for easy exportation of toxic compounds o Lac operon is regulated by a repressor protein § Lac operon can be transcriptionally regulated • By a repressor protein • By an activator protein § First method is an inducible, negative control mechanism • Involves lac repressor protein • Allolactose is the inducer o binds to lac repressor and inactivates in o Initiates transcription • When repressor is present RNA polymerase can bind to the promoter, but it cannot move up or down mRNA because the repressor is bound to the operator • lacI is constitutively expressed: always present • when there is no lactose in the environment, there is no allolactose o RNA polymerase cannot transcribe Z,Y and A genes o No Allolactoseà lac operon is repressed because operator is bound • Lactose present à some converted to allolactose à repressor released o Allolactose binding to the repressor causes conformational change that causes repressor to be unable to bind to the operator and the lac operon is transcribed o Lac operon is induced o When cell is exposed to lactose, some lactose gets in and some of that is converted to Allolactose o Allolactose causes repressor to fall off the operator sites o Cell can now metabolize lactose extremely well o Can be used for energy • most proteins from lac operon will be degraded overtime when there is no lactose and the cycle restarts (repressed à induced à repressed à ….) • Repressor does not completely inhibit transcription o Very small amounts of lac operon products will be in cell at all times à lac repressor is not 100% § Lac operon is also regulated by an activator protein • “catabolite repression” • When cell is exposed to both lactose and glucose, glucose is used first o Catabolite repression prevents the use of lactose when glucose is present o When glucose level is depleted, catabolite repression is lowered and the lac operon is expressed • Glucose causes very low cAMP levels in the cell, which prevents CAP from binding to initiate transcription • The sequential use of two sugars by a bacterium is called diauxic growth • When there is no glucose in the cell but only lactose, cAMP levels are high, so the CAP protein can bind to the CAP site and activate transcription because the repressor is not bound to the operator • Cyclic AMP binds to CAP dimer and initiates transcription if glucose is not present • Lactose presence inactivates the repressor • If neither glucose or lactose is present, repressor binds to operator o Repressor is stronger than activator • If glucose and lactose are present o CAP cannot bind to the CAP sites because cAMP levels are low o Repressor falls off of the operator because allolactose is present o transcription rate is very low because CAP cannot bind o small amounts of transcription • If glucose is present but lactose is not, only the repressor can bind to the operator o Transcription rate is extremely low § No CAP binding to activate transcription because cAMP levels are low § Repressor inhibits transcription - Trp operon is involved in the biosynthesis of the amino acid tryptophan o trpR: Encodes repressor protein § Functions in repression § Not a part of the trp operon o trpL: encodes leader peptide § Functions in attenuation § Part of the trp operon o Trp E,D,C,B,A all encode enzymes needed to synthesize tryptophan o Trp operon starts at the promoter and works all the way to the trpA o When low levels of tryptophan in the cell § Inactive trp repressor cannot bind to the operator site § transcription of the operon occurs o If tryptophan levels are high § Tryptophan binds to the repressor and changes its shape • Tryptophan is the corepressor here because it aids in repression § repressor conforms to new shape which can bind to the operator site and limit transcription o When attenuation occurs, transcription starts and only a short sequence is made before attenuator sequence is reached, where it is terminated § Occurs at a stop codon § There are two tryptophan codons within the stem loops § mRNA has four regions that can form stem loops that hydrogen bond with eachother • Second stem loop (3 and 4) is an intrinsic terminator (lots of uracil in this region) • Multiple loops are possible § Starting a promotor RNA is made, through the stop codon (passes through two tryptophan codons) § Region 1 and 2, and 3 and 4 can hydrogen bond together (2 can also hydrogen bond with 3 but not at the same time) § If the ¾ stem loop forms, transcription occurs only up to the u-rich attenuator sequence and then transcription stops § If no attenuation occurs, transcription of the rest of the gene continues § Transcription can occur without translation happening quickly behind it • this causes 1-2 and 3-4 hydrogen bonding to form stem loops • This causes attenuation • Most stable RNA is formed § If transcription and translation occur at the same time • Low tryptophan levels o there may not be enough tRNA charged with tryptophan, so ribosome stalls (right over region 1), allowing region 2 to hydrogen bond with region 3 o NOT ATTENUATED o Rest of tryptophan operon is translated • High tryptophan levels o ribosome stalls quickly on region 2 at the stop codon on TrpL gene, so region 3 and 4 hydrogen bond and form a stem loop à attenuation occurs o transcription stops - Inducible vs Repressible regulation o Operons involved in catabolism are normally inducible § Substance broken down acts as the inducer o Operons involved in anabolism are typically repressible § Inhibitor or corepressor is the small molecule that is the product of the operon - Riboswitches o RNA can exist in two different secondary conformation § One is active and the other inhibits gene expression § Conversions between the two forms is due to the binding of a small molecule § 3-5% of genes are regulated by riboswitches o Transcriptional regulation of TPP synthesis § If TPP concentration is low, one conformation exists (with stem loops) (transcriptional) • Can form antiterminator stem loop, allowing transcription to occur on the rest of the operon (thi operon, enzymes that are involved in TPP synthesis) § If TPP is high, TPP binds to RNA and affects its shape • forms a terminator stem loop • No transcription occurs and attenuation occurs • WE DON’T NEED TPP o Translational regulation of TPP synthesis • Low TPP has an exposed Shine-Dalgarno sequence (antisequestor), so ribosome can bind and translate RNA into a protein • If TPP is high, conformation switches and Shine-Dalgarno sequence is hydrogen bound to the start codon and other side of RNA o Ribosome cannot bind and translation cannot occur