Genetics Chapter 17 And 18

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Prokaryotic Gene regulation

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control of gene expression in a bacterial cell via mechanisms to increase or decrease the transcription and translation of specofoc genes and groups of genes

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induction

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process by which a signal induces expression of a gene or set of genes

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inducer

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small molecule that cause transcription from a gene or set of genes

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operon theory

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the theory suggested that a single signal can simultaneously regulate the expression of several genes that are clustered together on a chromosome and involved in the same process researchers reasoned that if a cluster of genes known as an operon will usually be transcribed together thus anything that regulates the resulting mRNA transcription will affect all genes in that cluster

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The Lac Operon

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Two environmental conditions have to be satisfied for the lactose metabolic enzymes to be expressed
1. lactose must be present in the environment
2. glucose cannot be present because glucose metabolism yeilds more usable energy
this is an example of catabolite repression
thanks to Francois Jacob, Jacques Monodthe metabolism of lactose requires a enzyme permease to transport lactose into the cell and B galactosidase to cleave the lactose molecule to yield glucose two genes encodes these enzymes all three genes are located on a signle mRNA RNA there is a fourth gene that encodes the lac repressor there is also a promoter site and a lac operator site

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Lac repressor

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the 5 segments on this mRNA represents a operon the interaction between the lac operator site on the DNA and the LAC repressor is crucial to the proper regulation of the lac operon the lac repressor contain two binding site one for DNA and the other to bind with a allosteric site that binds to analogs of lactose by binding to the operator the repressor prevents transcription by RNA polymerase when lactose of lactose analogs are present it can no longer bind to operaor

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Mutational analysis of the lac Operon

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Genetoc complementation testings in which we contruct bacterial genotypes heterozygous for various lac operon mutations can help distinguishing between different mutants. F' made it possible because ordinary bacteria are haploid but F' factors carrying the lac region can make them diploid and heterozygous for the the desired lac mutations

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inducible or constitutive??

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Wild type strains are inducible for B because in the presence of inducer enzymes are expressed but without the inducer in medium repressor in still binded no gene expression galactosidase and permease
mutations in the repressor and operator cause global misregulation of the lac operon strutural gene Oc mutations cause the lac operon to be constitutive meaning that regardless of whether the inducer is present the mutations lead to the transcription of the genes

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Cis acting repression

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Oc mutations are restricted solely to those lac structural genes on the same chromosome example O+Z−Y+/F′OcZ+Y− because the wildtype permease (Y+) gene is in cis to wild type operator permease activity is inducible in contrast the wild type b-Galactosidase (Z+) is in cis to the Oc mutant and therefore constitutive

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Trans acting repression

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IN I- mutants the DNA binding site of the repressor has been mutated so no functional operator binding protein is made wildtype I is inducible but mutants are constitutive and I is dominant to mut I. I+ is trans acting which means that the gene product regulates all structural lac operon genes

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Operon

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a unit of DNA composed of specific genes plus promoter and or operator that acts in unison to regulate the response of the structural genes to environmental changes

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The PaJoMo experiment

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they mated lacl- cell with a lacl+ LAcZ+ cell and they found that at first there was synthesise of B - Galactosidase but lacl+ accumulates synthesis stops and the addition of a inducer it will continue therefore lacI must be a repressor

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Positive regulation by cAMP how does it work

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transcriptional initiation at the lac operon is a comlex event in addition to the release of repression initiation also depends on a positive regulator protein that assist the polymerase in the start of transcription without it the helix will not open up very efficiently the CRP protein is a complex with cAMP is a positive regulator the binding between CRP and cAMP (Cyclic adenosine monophosphate) binds to the protein called cAMP high level expression of the lac operon requires that a positive regulator the CRP-cAMP complex be bound to the promoter region when little or no glucose is present adenyl cyclase activity is high and cause the synthesis of cAMP when glucose is high cAMP is low

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Loss of function mutations show that AraC is a positive regulator

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Expression of the arabinose genes in E coli requirres the AraC protein to bound next to the promoter. in a araC- mut the defective protein cannont bind and RNA polymerase will not transcribe the genes

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How was the lac repressor characterized

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molecular studies helped fill in the details of control mechanims 1866 Walter GIlbert purified the lac repressor protein and determined it was a tetramer of four identical lacI encoded subunits with each subunit containing an inducer binding domain as well as a domain that recognizes and bind to DNA they use radioactivity labeled repressor protein and a bacterial virus DNA that contained the lac operon to show that the repressor binds to operator DNA. When they combined the labeled protein to DNA and centrifuged the mixture in a glycerol gradient the radioactive protein co sediment with the DNA but the viral DNA lacOc did not

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Reporter gene

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a protein coding gene incorporated into a recombinant DNA molecule along with putative DNA regulatory elements after transformation of cells the reporter gene reports the activity of the putative regulatory elements to molecular biologists

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controlling gene expression using Fusions

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in addition to using lacZ as a reporter gene it can be fused to a regulatory region of gene X Expression will be dependent on signals in regulatory region in which it is binded too so how this is done is by ligating a DNA fragmentof the lac regulatory system in to a bacterial vector and you can control the expression of genes that are next to the lac gene regulatory system and the condition that induce lac expression will cause the expression fo growth hormone that can be purified from the cells

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catabolite repression

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repression of expression in sugar metabolizing operons like the lac operon when glucose or another prefeered catobolite is present

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Regulation of TRP operon

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E-coli tryp operon consist of 5 genes and is regulated by a protein repressor that is the product of the Tryp R gene unlike the lac operon who lactose functions as an inducer prevents it from binding when tryp is present it binds to the tryp repressor so it can bind to the operator and repress transcription interestingly enough Chaarles Yanofsky found that trp R mutants are not turned on sometimes and later found that two transcriptional outcomes can have different outcomes when tryp is absent ribosome stalls allowing normal stem structure to form when present ribosome moves quickly past codons allowing stem loop to form

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Sigma factors

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E Coli heat shock response depends on the the regulation of transcription by alternative factors these alternative factos which recognize different promoter regions ex. normal temp rpoH gene encodes sigma 32 has a promoter that regonizes sigma 70 but with high temperature there is no sigma 70 but recognized by sigma 24

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Cis acting elements

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Short DNA sequences 6-15 base pairs long that constitute the contol elements adjacent to genes through their binding to transcription factors cis control elements control or modulate transcription initiation at one or nearby genes exampples are enhancers and promoters

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Enhancer

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are cis acting sequences that can greatly increase transcription rates from promoters on the same DNA molecule they act to activate or positively regulate transcription silencers are cis acting sequence having reverse effects

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PRomoter region

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is a cis acting element that is very close to a genes initiation site

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Trans acting element

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a gene that encodes for a transcriptional factors that interact with cis acting elements either directly through DNA binding or indirectly through protein to protein interactions

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Different type of RNA Polymerase

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in Eukaryotes the finding that different classes of genes are transcribed by different RNA polymerases polI tandem repeats of rRNA genes are transcribed by RNA polymerase I transcribe one primary transcript PolIII recognize limited number of genes that encode small RNA molecules involved in the tranlation process Pol II recignizes promoters for proteins and micro RNA's

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String GENE

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all regulatory regions of the genome contains thousand of class II genes contain the promoter and an initiation site TATA box and and enhancer the string gene encodes a protein that activates the fourteenth mitosis of emobryonic development what is interesting is that cells in different areas or domains of the embryo enter at different times in an intricate but reproducible pattern

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Transcription Factors

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bind to an enhancer element can interact with the basil factors at the promoter to cause an increase in transcriptional initiation

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Activator

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a type of transcription factor that can bind to specific cis acting enhanver elements and increase the level of transcription from a nearby gene

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Dimerization domain

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a region of a polypeptide that facilitates interactions with other molecules if the same polypeptide or with other polypeptide

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the Myc-Max system

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is a regulatory mechanism for switching between activation and repression in a invitro experiment eventhough it contained leucine zipper and helix hoop helix it could not bind to DNA or form homodimers max can form homedimers and bind to DNA the max gene is expressed at all times but doesnt have a activator domain so max max homedimers when bound to the enhancer inhibit trasncription but MRx is not universally expressed so when expressed usually in cell going through cell prolifieration and MRx-Max dimers induces the expression needed for cell proliferation max max dimers repress proliferation

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locus conrol region

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a regulatory region first identified in the human beta globin locus but subsequently found in other loci the region is believed to regulate transcription by opening and remodeling chromatin structure it may also have enhancer activity human b globin gene cluster contains five genes in a cluster that can all be regulated by a distant LCR

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Chromatin structure in gene regulation

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-is the reduction of basal transcription from all genes to a very low level the main role of eukaryotic repressors is to modulate the activation of transcription caused by activators
-promoter regions are usually sequestered within the nucleosome and rarely bind to basal factors
-chromosomal regions nucleosomes have been eliminated

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micro RNA

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microRNAs (miRNA) are single-stranded RNA molecules of about 21–23 nucleotides in length, which regulate gene expression. miRNAs are encoded by genes that are transcribed from DNA but not translated into protein (non-coding RNA); instead they are processed from primary transcripts known as pri-miRNA to short stem-loop structures called pre-miRNA and finally to functional miRNA. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules, and their main function is to downregulate gene expression.

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Splicing factors of Sxl

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Sex lethal is an RNA splicing enzyme. Sex lethal's immediate target is Transformer mRNA. Since Sex lethal is not transcribed in males, its action on Transformer is restricted to females. Sex lethal acts positively in the functional splicing of Transformer mRNA. Transformer is another splice factor, acting in turn on downstream RNAs that require sex-specific splicing (Sosnowski, 1994). Transformer protein thus determines female developmental fate.

Act 2. In the spotlight here is dosage compensation, regulated by Sex lethal. The immediate target of SXL is male specific lethal-2 (MSL-2), a transcription factor. In the presence of SXL, MSL-2 is spliced into an inactive form, one that cannot function in dosage compensation. In the absence of SXL (in males), MSL-2 splicing is productive, and the active MSL-2 transcription factor effectively carries out dosage compensation.

A word about dosage compensation is in order. The ratio of sex chromosomes to autosomes in females (1:1) is different from the ratio in males (0.5:1). This is because males, by definition, have one X chromosome and not two. This presents a dosage problem. The ratio of gene products coded for by the sex chromosome will be different in males and females, unless some compensatory action is taken. The sex chromosome carries a lot of genes that are simply along for the ride, and the creation of a dosage imbalance spells catastrophe for development. What to do?