Regulation of Gene Expression AP Biology Ch 15

Gene Protein Control • Feedback inhibition – enough product is made the system shuts down – More product is made when needed – The product shuts down the process • Gene Expression – genes are only expressed when needed. Often regulated at transcription.

Gene Expression: Prokaryotes • Operon – grouped genes that are transcribed together – code for functionally similar proteins • Key Players – Promoter – section of DNA where RNA polymerase binds – Operator – Controls activation of transcription • on off switch • between promoter and genes for proteins – structural genes – Repressor protein – binds to operator to block RNA polymerase and shut down transcription • Turns off the operon • Corepressor – keeps the repressor protein on the operator – Trp operon • Inducer – pulls repressor off the operator – Turns on the operon – lactose on the lac operon – Regulatory gene – produces the repressor protein – Structural genes – code for proteins

Positive and Negative Gene Regulation • Negative • Positive – Repressible: usually on but can be inhibited trp operon, allosteric inhibition, tryptophan present prevents its own production. (anabolic) – Inducible: usually off, but can be turned on, an inducer (a specific small molecule, allolactose in the lac operon) inactivates the repressor and allows transcription (catabolic) – E. coli prefer to use glucose for energy, they will only use lactose when glucose is in short supply – glucose c. AMP binds to regulatory protein “CAP” & stimulates gene transcription Positive gene regulation! – The c. AMP & CAP combination allow RNA polymerase to bind to the promoter sequence more efficiently. – Remember c. AMP is regulating the gene expression in the bacteria

Trp operon: repressible, always making tryptophan, repressed if tryptophan is “eaten” tryptophan is necessary for the cell to function

Lac operon: inducible, only turned on if lactose is “eaten” lactose is not necessary for the cell to function

Eukaryotic Chromosome • Chromosomes – tightly coiled DNA around proteins during cell division • Chromatin – loosely packed DNA around proteins • Histones – protein which the DNA wraps around • Nucleosomes – grouped histones together – Heterochromatin – tighter packed chromatin • Not transcribing – Euchromatin – looser packed chromatin • Transcription occurring

Gene Expression: Eukaryotes • Cell Differentiation – cell specialization • All cells contain the same genes • The genes that are expressed determines the type of cell – Ex: Skin cell vs. a nerve cell

Chromatin Regulation • Histone acetylation – allows transcription factors to bind to DNA allowing transcription to occur – Creates loosely packed DNA - euchromatin • DNA Methylation – occurs after DNA synthesis has occurred – Lower transcription rates – One X in females is highly methylated – Works w/ a deacetylation enzyme in some spp.

Epigenetic inheritance • Not controlled by base sequences. • DNA methylation (deactivates one homologous chromosome) may explain abnormal or unexpected DNA expression as is often seen in identical twins. http: //images. the-scientist. com/content/images/general/55342 -1. jpg

Regulation of Transcription • Transcription involves RNA Polymerase II and transcription factors • RNA polymerase II attaches to the promoter (TATA box) sequence to begin transcription • Control elements – non coding sequences of DNA where the transcription factors attach

Regulation of Transcription. . AP Bio 15 -16Genetics15_10 Transcrip. Initiation_A. swf • Enhancer – control element far from a gene or intron • Activator – bind to enhancers to turn on transcription of a gene • Transcription factors + enhancer + activator + RNA Polymerase II = transcription initiation complex – Needed for transcription to begin • Repressors – inhibit gene expression – Turn off transcription – Block activators from binding to enhancers

Distal control element Promoter Activators Gene Enhancer TATA box General transcription factors 1 Activator proteins bind to distal control elements grouped as an enhancer in the DNA. This enhancer has three binding sites. 2 DNA-bending protein Group of Mediator proteins A DNA-bending protein brings the bound activators closer to the promoter. Other transcription factors, mediator proteins, and RNA polymerase are nearby. RNA Polymerase II Chromatin changes 3 The activators bind to certain general transcription factors and mediator proteins, helping them form an active transcription initiation complex on the promoter. Transcription RNA processing m. RNA degradation RNA Polymerase II Translation Protein processing and degradation Transcription Initiation complex RNA synthesis

RNA Processing Regulation • Alternative RNA Splicing – different regions of the pre-m. RNA serve as introns or exons creating different m. RNA strands depending on what is removed & spliced together.

m. RNA Degredation • Prokaryotes – Short Life span – Degraded in seconds – Allows rapid response to environmental changes • Eukaryotes – Survive from hours to weeks – Internal conditions constant, no need for rapid response

nc. RNA: 1000’s of RNA’s, current research • mi. RNA’s - micro RNA hat can degrade m. RNA or block translation • Causes m. RNA to fold on itself and base pair to create ds. RNA which is then digested with an enzyme • Short interferring RNA (si. RNA) – also degrade m. RNA or block translation (blocking by si. RNA is called RNAi, or RNA interferance)

Protein Degradation 18_12 Protein. Degradation_A. swf • Proteosomes – break apart proteins in to smaller peptide units Chromatin changes Transcription RNA processing m. RNA degradation Proteasome and ubiquitin to be recycled Ubiquitin Translation Proteasome Protein processing and degradation Protein to be degraded Ubiquinated protein Protein entering a proteasome Protein fragments (peptides)

Single Gene Expression • Different cells express different genes, therefore they make different m. RNA’s • We can detect m. RNA in a cell using nucleic acid hybridization, by pairing it to a nucleic acid probe • Each probe is labeled with a fluorescent tag to allow visualization • The technique allows us to see the m. RNA in place (in situ) in the intact organism and is thus called in situ hybridization

Figure 15. 16 Technique 1 c. DNA synthesis m. RNAs c. DNAs 2 PCR amplification Primers -globin gene 3 Gel electrophoresis Results Embryonic stages 1 2 3 4 5 6

Figure 15. 15 -5 DNA in nucleus 1 Test tube containing reverse transcriptase and m. RNA 2 Reverse transcriptase makes the first DNA strand. m. RNAs in cytoplasm m. RNA 5 Reverse transcriptase A A A 3 T T T 5 3 DNA Primer strand 3 m. RMA is degraded. 5 3 4 DNA polymerase synthesizes the second strand. Poly-A tail A A A 3 T T T 5 5 3 3 5 DNA polymerase 5 c. DNA carries complete coding sequence without introns. 5 3 c. DNA 3 5

Groups of Gene Expression • Recall Microarray assays: • Used to pinpoint differences in gene expression between 2 different cell types • How it’s done: – Sequence a genome – Use PCR to copy the genes (verification steps here) – Split the genes into single strands – Place the single stranded DNA onto microscope slides in spots (robots & computers do all this)