chapter 7- transcriptional control of gene expression

1. what are the two levels of Eu. transcription control?

1. chromatin level- open vs. closed 2. promoter level- DNA- binding transcription factors and other protein complexes assembled new gene promoters

promoter elements in E. Coli 1. most E. coli genes contain two promoter elements positioned ___ and ____ upstream of the transcription start site. 2. each element consists of a specific consensus* sequence ~6 to 10 bp long . What is a consensus sequence 3. correct _____ (distance) of the promoter elements is important for what? 4. one RNA polymerase molecule physically covers the entire region from -50 to +20 but the __________ makes sequence-specific contacts only with the -35 and -10 elements

1. -35 and -10 2. one that reps the most freq residues found at each position in a sequence element 3. recognition and binding of the RNA polymerase 4. one RNA polymerase molecule physically covers the entire region from -50 to +20 but the SIGMA FACTOR makes sequence-specific contacts only with the -35 and -10 elements

trans factor domains function independently 1. how many DNA- binding domains and activation domains are there in transciption factors? 2. DNA- binding domain determines which genes can be stimulated for trans but activation domains work when attached to any trans factor DNA- binding domain

1. 1 dna- binding domain and one or more activation domains.... the domains work independently

regulation of lac operon transcription 1. how many proteins does lac operon encode? _____ encodes B- galactosidase, the first gene in the operon. 2. what are the 3 distinct regions controlling transcription? 3. when ___ is absent and ___ is present, there is no need for the cell to metabolize lactose. so transcription of the lac operon is prevented how? 4. what about when there's lactose and no glucose? 5. what about when both are present? 6. when there's no glucose, how is more transcription stimulated?

1. 3; lacZ 2. promoter, cap site, operator (overlaps start site) 3. lactose is absent and glucose is present to prevent transcription( cos its basically just a waste), the lac operon binds to the start site/ operator region and prevents binding . no lactose but having glucose means that there are low levels of cAMP which serves as a signal 4. whens there's lactose, then it'll bind to the lac repressor causing a conformational change and dissociation from the operator. 5. when both are present, its low transcription cos some repressor is still bound. 6. when theres no glucose, cells make cAMP which binds to CAP, forming cAMP-CAP complex which binds upstream = HIGH transcription

RNA Pol II promoter elements: initiator sequences 1. This class of promoters contains an _______ that ______ the start site. 2. The initiator sequences are ________ compared to TATA box

1. Initiator sequence that overlaps 2. Poorly conserved

leucine zipper trans factors 1. many proteins, including trans factors contain alpha- helices with the hydrophobic aa _____ at every 7th position and form ________ 2. since α-helices have ~3.6 residues/turn, leu at every 7th position makes one side of each helix ___________ (amphipathic helix) facilitating their coiling around each other to form a _______ (like a zipper) 3. basic aa's at one end of the ______ bind to specific sequences in adjacent ________ like a pair of scissors

1. Leucine at every 7th position and form coiled- coil dimers 2. one side of each helix hydrophobic facilitating their coiling around each other to form a dimer 3. basic aa at one end of the dimer bind to specific seq in adjacent major groove

structural features of activation/ repressor domains in trans factors 1. many activation domains are rich in _____ amino acids. 2. some activation domains, esp acidic ones, remain unstructured ones (_______________) until they bind to a _________________. 3. some activation domains are highly structured but require binding to what in order to change to an active conformation?

1. acidic aa 2. unstructured as in they do not fold into a specidc tertiary structure until they bind to a co-activator protein. 3. binding to a ligand --locks it into the right conformation.

DNA- binding proteins control gene transcription 1. proteins that bind to promoters and other control regions act as _____ or _____ of gene trans. 2. these regulator proteins recognize and bind to ______ in promoters and other control regions and are called _____ (usually 6 to 10 bp long) 3. what do repressor proteins do? 4. what do activator proteins do?

1. act as repressor or activator 2. short specific DNA sequences, promoter elements 3. repressor- prevent binding of RNA poly at the start site or prevent it from transcribing activator- enhance recruitment of RNA poly at start site or stimulate the rna poly thats already bound.

RNA Pol II promoter elements: TATA Box 1. where is it commonly found? 2. where is it located in terms of the start site?

1. commonly found in highly transcribed genes 2. 25 to 35 bp upstream of start site, the TATA box contains consensus sequence thats 8 nt long

activation of estrogen nuclear receptor by ligand binding 1. estrogen binding to the activation domain of the estrogen receptor causes a ___________ that allows activation of pol II via a co-activator protein. 2. when a drug for ex, tamoxifen, binds to the same site as estrogen it blocks the required conformational change and thus is a ________.

1. conformational change (agonist) 2. antagonist

homeodomain trans factors 1. DNA- binding domain is a _______ sequence ~60 aa long and is similar to helix- turn- helix motif of bacterial repressors 2. homeodomains are most often found in transcription factors that function during _______

1. consensus 2. development

modular nature of transcription factors 1. trans. activating functions and dna binding act. reside in ______ of the protein 2. yeast GAl 4 trans. factor binds to the UAS gal control element found in genes required for metabolizing galactose 3. ________ of the GAL 4 protein can be assayed for binding to UASgal and for stimulation of a reporter gene introduced into yeast cells

1. different domains 3. deletion constructs -- also since activating func and dna binding func are at different ends where you cut the protein also determines where there will be binding as well.

principles of dna- binding specificity in transcription factors 1. mos bacterial repressors and many eukaryotic transcription factors bind as ______. 2. bacterial repressors ontain a DNA- binding domain called _______ 3. what determines DNA binding affinity?

1. dimers 2. helix- turn- helix motif 3. specific contact between amino acid side chains in the recognition alpha- hellix (sequence reading helix) and bases in the major groove of DNA

1. distant control elements called _________ sequences often stimulate transcription. 2. using 50 to 200 bp long and consist of multiple individual ____________, which are positioned close to each other ( an ________ binds multiple transcription factors) 3. ______ stimulate trans of any gene even when present _______ from the start site (upstream, downstream within introns, or even after the last exon) 4. often responsible for _______- specific trans of gene

1. enhancer 2. control elements, enhancer seq 3. enhancer, thousands of bp away 4. tissue- specific

RNA I, II, III subunit comparison (remember these are the ones involved in Eu. gene transcription) 1. All 3 Eu. polymerases have how many core subunits (out of the 12-16) homologous to bacterial counterparts? 2. only RNA pol II (copies mRNA) has a _________ in its largest subunit. 3. what is the role of pol II CTD phosphorylation?

1. five 2. C- terminal domain (CTD) 3. it helps initiate mRNA transcription and is required for recruiting enzymes for mRNA processing (capping, adenylation, splicing, etc)

mediator complex is essential for pol II activation by transcription factors 1. the large subunit co- activator complex called _______ forms a bridge between activator domains of transcription factors and pol II

1. mediator

yeast genes are simpler: 1. how many control elements does it have?

1. most contain only one upstream DNA control element, called an upstream activating sequence (UAS)

transciption facot act. is often regulated 1. what are the two ways Eukaryotes can respond to extracellular signals?

1. one way is thru the transmembrane receptors through a reaction cascade --> leading to activation or repression of transcription factors already present 2. a second way is through lipid-soluble molecules (aka nuclear receptors) that diffuse thru the membrane and DIRECTLY activate trans. factors by binding to and changing the comformation of the activation domain

pausing of pol II transcription and regulation by elongation factors 1. at most promoters, pol II _____ after transcribing ~20-50 nts due to binding of a protein complex called ________. 2. elongation proceeds following binding of another factor ____, and _____ of the pol II CTD domain as well as phosphorylation of NELF and DSIF

1. pauses, NELF- negative elongation factor 2. DSIF, phosphorylation

testing putative transcription factor activity by transfection 1. there are two plasmids... what would plasmid 1 contain and what would plasmid 2 contain? 2. ____________ of the two plasmids in cells lacking X protein increases reporter gene expression if the transcription factor is an ________ or decreases expression if it is a _________

1. plasmid 1 would have the transcription factor and plasmid 2 would have the control element as the binding site and a reporter gene with a minimal promoter like a TATA box. 2. co-transfection of the two plasmids in cells lacking X protein increases reporter gene expression if the transcription factor is an activator or decreases expression if it is a repressor (transcription factors can be either activators or repressors)

transcription control at the promoter level 1. where are promoter regions and they serve as binding sites for what? 2. promoter regions are _____ for gene transcription. 3. the promoter region govern the ____ of a gene and its response to regulatory signals. ( what binds to the site determines how much)

1. promoter regions are upstream of gene transcription start sites. binding site for RNA polymerase and regulatory proteins 2. promoter regions are REQUIRED 3. govern the TRANSCRIPTION LEVEL

electrophoretic mobility shift assay (EMSA) 1.useful for detecting transcription factors during ______. 2. column chromatography is used and the column fractions are then incubated with _______ DNA fragment containing a ___________ (probe) followed by gel electrophoresis. 3. when a transcription factor binds to the control element, a ______ migrating protein- DNA complex is observed.

1. purification 2. radiolabeled; control element (probe) 3. slower

RNA pol II promoter elements 1. in Eu. what does "promoter"/ "core" promoter refer to? 2. Core promoter elements recognized by pol II fall into 3 main classes which are?

1. refer to those DNA control elements that are close to the start site and recruit and position RNA poly II for transcription initiation. 2. TATA boxes, initiator sequences, CpG islands

1. some trans factors have _______ in place of activator domains and thus inhibit rather than activate gene transcription 2. what do mutations within transcription factor repressor domains often lead to?

1. repressor domains .. they alos work independently 2. they often lead to constitutive (unregulated) expression of the genes they control

1. DNA binding sites (control elements) for nuclear receptors are referred to as __________. 2. response elements either contain _______ repeats or ______ repeats and bind _______ of nuclear receptor proteins. 3. different response elements, depending on location (cytoplasm or nucleus), it'll either repress or promote.

1. response elements 2. direct or indirect repeats and bind dimers

SIigma factor are essential for binding of E.coli RNA poly to promoters 1. what are the sigma protein factors responsible for? 2. transcription of most genes in exponentially growing E. coli cells require what sigma factor?

1. sigma factors assoc with the rna polymerase and recog the -10 and -35 promoter elements before the rna poly bind to the promoter 2. sigma factor 70

1. DNA- binding proteins of eu. transcription factors can be grped into related structures with ______________. what are the 3 best known structural types?

1. similar motifs 3 best known: homeodomain, zinc finger, leucine zipper

alternate sigma factors in e. coli 1. alternate sigma factors are used for gene activation in response to environmental signals. genes that require alternate sigma factors generally control a _______. 2. how many sigma factors does E. coli have?

1. specific function 2. it has 7 sigma factors, and each factor recognizes a specific consensus sequence

transgenic mice 1. To make transgenic mice, ______ containing any gene(s) of interest is simply ______________. 2. multiple copies of the added DNA insert randomly into host chromosomes by nonhomologous recombination

1. to make transgenic mice, foreign DNA containing any gene(s) of interest is simply injected into fertilized eggs. 2.

Eu. gene transcription reg= MANY MORE DNA CONTROL ELEMENTS 1. what are transcription factors? 2. transcription of a single gene is regulated by what? 3. different cell types accumulate a specific set of _______ that bind to a specidic _______ ( a major factor in determining which genes are expressed)

1. transcription factors are repressor and activators of transcription that bind to DNA control elements in eukaryotes. 2. multiple DNA control elements and multiple transcription factors 3. specific set of transcription factors that bind to a specific control element.... very specific!!

reg. of transcription elongation in bacteria expression of some bacterial genes is also regulated at the elongation stage of transcription-- tryp operon 1. when tryptophan is present as a nutrient, how is transcription reduced? 2. how is further reduction of tryp operon proteins acheived? 3. how is this mediated-- what is formed?

1. transcription reduced by tryp repressor protein 2. its achieved by preventing elongation of the transcript -- called attenuation 3. mediated by alternative base- paired structures --> termination

regulation of gene transcription in bacteria is relatively simple 1. promoter region upstream of gene starts are small ( <50 bases long); how many promoter elements do they usually have? 2. How is the core RNA poly. positioned for the correct start site? 3. how many regulator proteins does the promoter region generally bind (repressors and/ or activators)?

1. two or three 2. it has to associate with a promoter- recognizing subunit called sigma before it can bind to the specific DNA promoter element. 3. one or two

abundance of specific transcription factors 1. humans encode lots of transcription factors!! each trans factor binds to specific control element 2. transcription level for each gene results from the combined effects of what?

2. combined effects of multiple transcription factors bound to their control elements

zinc finger transcription factors 1. zinc fingers motifs are very common in transcription factors 2. zinc comes first and binds protein then => zinc finger motif... _______ and ________ in the motifs (either C2H2 or C4) bind zinc ions via their side groups

2. cysteines and histidines ... the binding to zinc fingers allow struture of protein to bind to particular seq

detection of specific trans factors in cell extracts: footprinting assay 1. incubate protein extract with a radiolabeled DNA frag containing a putative control elements ( only 5' end is tagged) 2. add small amounts of endonuclease DNase I to produce about one random cut/ DNA frag --> if the trans factor that can bind to the control element is present in the protein sample, the site is _______ from cutting by the endonuclease. if it was incubated without protein prior to DNase I digestion, the gel electrophoresis resylts in an array of bands reflecting _________ cutting

2. protected , random cutting

1. 60-70% of protein encoding genes aren't transcribed as frequently as the ones containing a TATA box or an initiator seq (ex - many housekeeping genes required for basic metabolic processes), these genes utilize a _________ promoter sequence (GC rich regions)

CpG

In eu. gene transcription, there are ___ RNA polymerases involved which is made up of how many subunits? how subunits is bacterial RNA polymerase made up of?

there are 3 RNA polymerases... Eu. structure made up of 12-16 subunits and bacterial RNA poly made up of 5 subunits.