BioLec120109.doc

Bio 101 Lecture Notes for December 1, 2009: Exception: RETROVIRUSES use RNA to synthesize protein All genes are not always being transcribed in all cells. CONSTITUTIVE GENES: always being expressed in all cells products are essential for normal cell growth and viability AKA ?housekeeping genes? REGULATED GENES: expression is controlled in response to needs of cell / organism products are needed at specific times or in certain cells or in the presence of certain nutrients lecture today will focus on how they know when they should be expressed Controlling Gene Expression in Prokaryotes: Sidenote: The ?promoter? is the DNA sequence that RNA polymerase will bind to before it starts to transcribe Mainly done by turning transcription on / off at the right time A well-studied example: the lac operon. OPERON is a cluster of genes coordinately regulated by one promoter; they are only found in prokaryotes [figure 14-2] The lac operon: Studied by Jacob and Monod (1961) Contains structural genes that metabolize lactose in E. coli. [Structural genes code for polypeptides; structural genes are genes that are transcribed into a message and then translated into a protein] E. Coli metabolizes lactose this way: lactose ([enzyme beta-gal breaks it down into two separate sugars]( glucose and galactose ( energy Protein products: beta-gal, permease, transacytlase Jacob and Monod observed: E. coli grown on media containing LACTOSE had LOTS OF BETA-GAL ACTIVITY. E. coli grown on media containing GLUCOSE had nearly NO BETA-GAL ACTIVITY Their results showed that E. coli only express beta-gal when they need to. Only when the substrate is available are the making what?s necessary. E. coli only express lac Z, lac Y. lac A genes when cells need protein products (in the presence of lactose) This is induction Induction: turning on of gene expression Inducer: environmental agent that triggers induction (in the case of lac operon, it?s lactose) How does the presence / absence of lactose in the environment regulate gene expression inside bacterial cells? It does so by the repressor gene. The REPRESSOR GENE is not part of the operon; it?s always expressed because it?s a constitutive gene. The repressor gene codes for repressor protein. The repressor protein binds to the operator; when it?s bound to the operator, then transcription cannot happen. So the repressor protein prevents transcription in the absence of lactose. In the absence of lactose: Repressor protein is bound to operator DNA sequence Repressor protein physically blocks RNA polymerase No transcription of lac Z, Y, or A. When lactose is present in the media: Lactose is transported into the cell and converted into allolactose Allolactose binds to Repressor Protein Now Repressor cannot bind DNA RNA Polymerase is not blocked Lac z y and a are all transcribed Polycistronic mRNA is translated [Polycistronic mRNA: one message has coding sequence for multiple protein products; only in prokaryotes] ?. [So the repressor protein sees the level of lactose present and this allows the repressor protein to be the operator] Repressor protein negatively controls expression of lac operon. Negative control: regulatory protein turns OFF transcription; transcription is ON only when repressor is not active (cannot bind DNA) ? Lac Operon is also POSITVELY REGULTED by Catabolite Activator Protein (CAP). Positive regulation: regulatory protein turns ON protein; active CAP binds at 5? end of promoter to help RNA Polymerase bind. CAP needs cAMP to be active So we have two specific DNA binding sites: the CAP binding site and the operator. cAMP is only made when there is low glucose in the cell. Glucose is the preferred substrate for bacteria. It?s easier for the bacteria to break down glucose than to break down lactose CAP allows cell to determine if glucose is present. Glucose is a simple sugar and easier to metabolize than lactose. If bacterial cells are grown in lactose AND glucose: glucose will be used; there?s no need to transcribe lac Z, lac Y, or lac A. BUT, the repressor is not blocking RNA Polymerase. SO, presence of inactive CAP prevents transcription. In this situation, we?ll have high level of glucose which means no cAMP and therefore, no active CAP. If bacterial cells are grown in lactose WITHOUT glucose: lactose must be used; low glucose(lots of cAMP(active CAP; active CAP turns on transcription of lac Z, Y, and A by helping RNA Polymerase bind promoter ***Summary of how bacteria sense levels of glucose: high glucose ( no cAMP bindingto CAP [because the presence of glucose inhibits the enzyme that makes cAMP] so no active CAP; no binding to DNA; inefficient RNA Polymerase; no transcirptino of lac genes. If glucose is low, cAMP is present, and binding of cAMP and CAP will occur; so binding to DNA does happen and RNA Polymerase binding is efficient; that there is a transcription of lac genes. ***Important players in this process: PROMOTER: site on DNA where RNA Polymerase binds; [promoter also has the CAP binding site] OPERATOR: site on DNA where Repressor protein binds GENES: lac Z (codes for beta-gal, which breaks down lactose); lac Y codes for permease; lac A codes for transacetylase REPRESSOR GENE: not part of operon; product = Repressor protein ***Summary of Regulation of gene expression at the lac operon: In the absence of lactose: repressor protein binds at operator; no transcription because RNA polymerase is blocked In the presence of lactose: lactose is made into allolactose; allolacotse binds to repressor protein; now repressor protein cannot bind to operator; RNA polymerase transcribes lac Z, Y, and A; no glucose means high cAMP, so active CAP to help RNA Polymerase bind In the presence of lactose AND glucose: lactose is converted into allolactose; allolactose binds to repressor protein; now repressor protein cannot bind at operator; high glucose means low cAMP, so no activeCAP; RNA Polymerase cannot stably bind, so no transcription of lac Z, Y, or A. IN EUKARYOTES: Controlling Gene Expression in Eukaryotes: No operons in eukaryotes All genes have their own promoters More levels of control: DNA packaging / turning transcription on or off / post-transcriptional? DNA PACKAGING: Specific folding of DNA by histone proteins [histone proteins have a net positive charge which allows them to interact with the negative charge in the DNA] Basic unit = nucleosome. [NUCLEOSOME: DNA wrapped around 8 histone proteins] Can be highly condensed or more relaxed [figure 14-7]. When highly condensed: nucleosomes fold up to form 30 nm fiber; tight packing; no transcription; HETEROCHROMATIN; example: Barr bodies. When relaxed: allows for transcription; EUCHROMATIN Regulating gene expression by TURNING TRANSCIRPTION ON OR OFF: [fig 14-9] Promoter sequence is important. Promoters are much more complex in eukaryotes. Promoters include TATA box + Upstream (of the TATA box) Promoter Elements (UPEs) [Remember that G and C form 3 bonds. A and T form 2 hydrogen bonds]. Different numbers and types of UPEs regulate different levels of transcription. Promoter sequence is bound by specific proteins (transcription factors). COORDINATED GENE REGULATION IN EUKARYOTES: Several eukaryotic genes can be activated by the same stimulus. Stimulus: cellular stresses, hormones, nutrients, development, etc. In this case, EUKARYOTIC GENES all have the same UPEs in their promoters. Individual eukaryotic genes can be activated by different stimuli. EUKARYOTIC GENES here have unique combo of UPEs. Mix of regulatory elements in promoters varies: diff combos of elements; different copy numbers of each element present (slide from last class) PRE-mRNA PROCESSING: post transcriptional control: 3 steps( [1] 5? capping; [2] 3? Poly (A) tail; [3] ____ splicing Post transcriptional control implies alternative splicing. REGULATING ACTIVITY OF NEWLY MADE PROTEIN: Post-translational control Examples of ways this is done: chemical modification (phosphorylation, acetylation, etc.); precursor cleaved to generate active protein ***SUMMARY: regulating gene expression in pros versus euks PROs: regulation mainly by turning trans on or off Coordinate gene expression due to operons EUKs: many level of regulation: DNA packaging; turning trans on or off; postranscirptional control; post-translational control Coordinated gene expression is due to common UPEs [and not operons]