Module 8 Flashcards
Gene Regulation
The ability to alter the expression of genes
Gene regulation can occur at three distinct points in bacteria:
- Transcription
- Translation
- Posttranslation
Regulatory Proteins
Utilized in transcriptional regulation, includes repressors (bind to DNA and inhibit transcription) and activators (bind to DNA and increase transcription) and influenced by small effector molecules
Small Effector Molecules
Bind to regulatory proteins, influence whether or not they can bind (inducers and corepressors)
Inducers
Increase transcription by either binding to activators or binding to repressors and preventing them from binding
Genes regulated by these small effector molecules are “inducible”
Corepressors
Decrease transcription by either binding to repressors or binding to activators and preventing them from binding
Genes regulated by these small effector molecules are “repressible”
Enzyme Adaptation
A particular enzyme appears in the cell only after the cell has been exposed to the enzyme substrate
Operon
Regulatory unit consisting of a few protein-encoding genes under the control of a single promoter, encodes a polycystronic mRNA sequence
DNA elements that control lac operon transcription:
- Promoter (binds to RNA polymerase)
- Operator (binds to lac repressor)
- CAP site (binds the Catabolite Activator Protein)
lac Operon Structure
i promoter > lacI regulatory gene > CAP site > lac promoter > operator site > lacZ > lacY > lacA > lac terminator
lacZ Gene
Encodes B-galactosidase which enzymatically cleaves lactose, converting into allolactose
lacY Gene
Encodes lactose permease, allows for transport of lactose across cell membrane
lacA Gene
Encodes galactoside transacetylase which acetylates lactose to prevent buildup of toxins
After B-galactosidase breaks down lactose into allolactose, it acts as an ____ and binds to ____, preventing it from binding to the DNA
inducer, lac repressor
increases transcription
lacI Gene
Encodes the lac repressor, which binds to the operator when no lactose is present
Jacob, Monod, and Pardee Experiment Results
Determined that merozygote (lacI+ on chromosomal, lacI- on F’ factor) will remain inactive when lactose is absent and activate in presence of lactose
Due to presence of active repressor protein in chromosomal gene
Loss of function in lacI vs lacO
When negative with no lactose, both are at 100% expression. With lacI+ on F’ factor both will express 200% when lactose is present, when it is not present lacI will then cut its expression to 0% while lacO will still produce 100%
Catabolite Activator Protein (CAP)
In the presence of two sugars (diauxic growth), bacteria use glucose before lactose. Glucose inhibits cAMP production and leads to lack of small effector molecules needed to start lac operon
Results of lac operator research
Discovery that lac operon has three separate activators (O1 next to promoter, O2 downstream within lacZ gene, and O3 slightly upstream of promoter)
Found that DNA loops form from binding of lac repressors; if not all bound, O1 must bind to either O2 (stronger repression) or O3 (weaker repression)
Binding of lac operators (O1 to O2 or O1 to O3) forms a ____ since operators are far apart
loop
Riboswitches can regulate transcription and translation via the formation of a ____
stem loop (terminator/anti-terminator in RNA for transcription, inhibits translation of Shine-Dalgarno RNA sequence in translation)
Gene Regulation in Eukaryotes
Can occur at any point in the transcription, RNA processing, translation, posttranslational modification process
Combinatorial Control
Most eukaryotic genes regulated by many factors, such as activators and repressors
Activators and repressors in eukaryotes can be modulated by ____
small effector molecules, protein-protein interactions, covalent modifications, also via regulatory proteins, DNA methylation, and formation of heterochromatin
Activators and repressors bind ____ of the primary promoter
upstream
Regulatory transcription factors control ____ either directly or through coactivators to enhance transcription or silence it
TFIID
Glucocorticoid Pathway
- Glucocorticoid molecules enter cell via glucocorticoid receptor
- Receptors binds to Glucocorticoid Response Element (GRE) inside cell
- GRE then positions itself on the chromatin and functions as an enhancer for many genes
CREB Protein Pathway
- cAMP increases in response to extracellular signaling
- CREB binds to two adjacent sites (cAMP response elements - CRE)
- CREB then binds to specific binding protein, CBP, which also serves as a coactivator and causes transcription to dramatically increase
ATP-Dependent Chromatin Remodeling
Dynamic changes in chromatin structure which repositions and restructures nucleosomes, goal is to make DNA more or less open for transcription
Histone Variant
Histones that have been altered through accumulated mutations, specialized to certain cell types or genome regions
Histone Code
Modifications of amino terminal tails of histones can affect transcription by influencing interactions between DNA and histone proteins and by occurring in patterns recognized by proteins
ChIP-Seq Technique
Technique used to map locations of specific nucleosomes within genome, allows for the determination of nucleosome/histone variant locations and where histone modifications occur.
- After breaking DNA into segments, Abs specific to histones and attached to heavy bead bind to them
- Centrifuged which produces pellet with core histones and cross-linked proteins
- Exposed to agent to separate histones from DNA before sequencing to determine where sequence originates from
Nucleosome-free regions are found at the beginning and end of most genes
When a gene is transcriptionally activated, chromatin remodeling and histone modification occurs via ____
remodeling complex, histones are evicted and acetylation occurs
DNA Methylation
A covalent attachment of methyl groups carried out by DNA methyltransferase, often inhibits transcription
Methylated DNA sequences are inherited during cell division (genomic imprinting). Specific methylated genes from mother and father via ____, and maintained in offspring via ____
de novo methylation, maintenance methylation
A small effector molecule induces transcription. Which combination below is consistent with this idea?
A. Repressor and inducer
B. Activator and inhibitor
C. Repressor and corepressor
D. None of the above
A. Repressor and inducer
The inducer prevents the repressor from binding, allowing transcription to move ahead
Let’s suppose that mediator has interacted with an activator protein. What would you expect to happen next?
A. TFIID would bind to the core promoter
B. RNA polymerase would bind to the core promoter
C. Histones would be displaced from the core promoter
D. RNA polymerase would switch to the elongation phase of transcription
D. RNA polymerase would switch to the elongation phase of transcription
RNA polymerase is already bound to the core promoter, however mediator lies in its path along the operon preventing transcription
If histones were not removed during eukaryotic gene transcription, which processes would be inhibited?
A. The formation of the pre-initiation complex
B. Transcriptional elongation
C. Phosphorylation of CTD
D. Both A and B
D. Both A (The formation of the pre-initiation complex) and B (Transcriptional elongation)
Which of the following phenomena could you study using ChIp-Seq?
A. To see if a regulatory transcription factor binds to mediator and/or TFIID
B. To measure the level of transcription of a given gene in real time
C. To analyze the binding of protein(s) to DNA on a genome-wide scale
D. To determine if a mutation at a regulatory site alters the transcription level of a given gene
C. To analyze the binding of protein(s) to DNA on a genome-wide scale
