Gene Expression and Its Control
Explain the regulation of the lac operon
+ Glucose/ +Lactose = OPERON OFF because CAP not formed + Glucose/ - Lactose = OPERON OFF because lac repressor bound and CAP not formed - Glucose/ - Lactose = OPERON OFF because lac repressor bound - Glucose/ +Lactose = CAP and cAMP enable RNA polymerase to function No Lactose = repressor bound No Glucose = CAP bound
What are the key points regarding DNA motifs?
1) DNA binding and activation region exist as "independent" domains 2) Specific interactions with major/minor groove of DNA 3) In many cases can bind as dimers 4) Binding can be co-operative 5) Can be categorized into "classes" based on their DNA binding function, but also on their overall "function" in transcription
What are some ways that transcription factors are activated?
1) Ligand Binding (addition of ligand) 2) Protein Phosphrylation (addition of phosphate) 3) Addition of Second Subunit (addition of DNA binding subunit) 4) Unmasking (removal of inhibitor-phosphate complex) 5) Stimulation of Nuclear Entry (removal of inhibitor protein) 6) Release from Membrance 7) Protein Synthesis
What is RNA splicing?
1) Transcription, 5' capping 2) Cleavage at poly(A) site 3) Polyadenylation 4) RNA splicing These steps can control the expression level of a particular gene. 15% of all human genetic disorders result from splicing defects
What are the forms of protein expression regulation?
1) Transcriptional control 2) RNA processing control 3) RNA transport and ionization control 4) Translation control 5) mRNA degradation control 6) protein activity control
Why cap and add poly-A tail to mRNA?
1) stabilizes the mRNA transcript, protect the ends of the transcript 2) helps with export of mRNA out of nucleus 3) enhances the efficiency of translation
What are strategies employed in transcriptional repression?
A) Competitor DNA Binding - Repressor can get in the way of the activator B) Masking the Activation Surface -- Repressor can bind to the activator C) Direct Interaction with General Tx Factors -- Enhancer and the silencer are separate. Activator does not work when the repressor is bound to the silencer.
What are the four functional classes of transcriptional activators?
A) DNA-bound activators that recruit additional protein partners required for transcription, B) co-activator proteins that modify the arrangement of chromatin, C) the mediator/bridging complex, and D) general/basal transcription factor proteins.
What are common activation domains?
Acidic domains Glutamine-rich domains Proline-rich domains Activation domain allows the recruitment of other proteins that ultimately facilitate the binding of RNA polymerase to the promoter, and formation of the pre-initiation complex (PIC). The activation domain of some co-activators contains enzymatic activity.
What are the transcription factors involved in transcription initiation by RNA polymerase II in a eukaryotic cell (A-D)?
Activator proteins (A) help recruit and assemble coactivators (B), the mediator complex (C), and RNA polymerase and the general factors (D), at the promoter. Proteins bind to specific short sequences in DNA (cis elements)
Explain the chromatin remodeling process of transcriptional activation
Activators recruit Co-Activator proteins required for Chromatin Remodeling Step 1: Activator binds to the cis element. Recruits non-DNA binding co-activator which causes DNA sliding (ATP dependent) and increases access to DNA promoter (purple). Step 2: Histone modification (acetylation) further increases access. Step 1 is energy dependent. Step 2 can lead to recruitment of other factors that only recognize acetylated DNA.
What factors are assembled at the promoter to activate transcription?
Activators, co-activators, basal transcription factors
What are agonists?
Analogues that bind to a receptor and trigger signaling Tissue specific agonist/antagonist: Raloxifene is an estrogen receptor agonist in bone, but an antagonist in breast (SERMs)
What are antagonists?
Analogues that bind to a receptor but fail to activate it Ex: Tamoxifen binding to the estrogen receptor (ER). Tissue specific agonist/antagonist: Raloxifene is an ER agonist in bone, but an antagonist in breast (SERMs)
Illustrate how transcription factors generally possess multiple and independent domains, and define the functional significance of this architecture.
Basal levels of transcription for most eukaryotic genes are low. Efficient transcription depends on the presence of activator proteins that have independent DNA-binding domains and activation domains. The DNA-binding domain allows binding of the activator to specific enhancer sites upstream or downstream of the promoter. The activating domain "recruits" other proteins needed for transcription. Among the most common DNA-binding domains are the zinc finger domains, helix-turn-helix, leucine zipper, homeobox domains, and basic helix-loop-helix structure.
Explain the function of tamoxifen
Blocks the ability of estrogen receptor to become active thus blocking growth of certain estrogen-dependent breast cancers. Binding of tamoxifen to estrogen receptor blocks interaction with coactivator
What are the key points about RNA processing?
Capping, polyadenylation and splicing can all control the levels of RNA being synthesized Five snRNPs interact with each other and with the pre-mRNA to form the spliceosome The spliceosome catalyzes 2 transesterification reactions and joins together 2 exons Alternative splicing is the primary mechanism for regulating mRNA processing 15% of all human genetic disorders result from splicing defects
What are leucine zippers?
Consists of a stretch of amino acids with a leucine residue in every seventh position. A leucine zipper in one polypeptide interacts with a zipper in another polypeptide to form a dimer (cooperative binding). Adjacent to each zipper is a stretch of positively charged residues that is involved in binding to DNA.
What is Rett's Syndrome?
Developmental disorder (mostly females) Mutation in the Methyl-CpG-binding Protein 2 (MeCP2) gene of the X chromosome. The MeCP2 protein binds mainly to methylated DNA (CpG dinucleotides) and recruits co-repressor complexes containing histone deacetylases that are able to alter chromatin structure to inhibit gene expression during neurological development.
What is alternative splicing?
Different exons are combined to make unique proteins in different tissues (e.g., beta-thalassemia mutations)
What is an enhacesome?
Eukaryotic gene regulatory proteins assembled into large complexes
What are anabolic steroids?
Excessive use in men causes decreased secretion of testosterone, testicular atrophy, and sometimes breast enlargement. In women, excessive testosterone decreases ovulation and estrogen secretion and growth of facial hair.
What is a splicesome?
Five snRNPs interact with each other and with the pre-mRNA to form the spliceosome The spliceosome catalyzes 2 transesterification reactions and joins together 2 exons
What is euchromatin?
Less condensed, transcriptionally active (Eu = true, "truly transcribed"; Eu are on)
Discuss the phenotype and treatment for rickets
Low calcium Alopecia Rx: calcium infusion
What does the Metallothionein protein do?
MT binds metals as well as OH free radicals. The proteins that bind to the cis-elements in the promoter region of the MT gene are the "signal transducers". The promoter contains many different cis elements so that multiple signal transduction pathways can "converge" and control the transcription of this MT gene.
Where are enhancers and silencers located?
May be close to, far from, or even within (in an intron) the gene whose expression it regulates.
What are basic helix loop helix?
Motif has been identified in some eukaryotic DNA-binding proteins. An amphipathic helix presents a face of hydrophobic residues on one side and charged residues on the other side. The length of the connecting loop varies from 12-28 amino acids. The motif enables proteins to dimerize, and a basic region in the DNA binding helix inserts in the major groove of DNA.
What is the helix-turn-helix motif?
Motif was originally identified as the DNA-binding domain of phage repressors. One α-helix lies in the major groove of DNA; the other lies at an angle across DNA.
Describe the Wilm's tumor gene (WT1)
Mutated maternal/paternal WT1 genes -- no functional protein -- kidney tumors The WT1 protein, which has a C2H2 zinc-finger DNA-binding domain, binds to the control region of the gene encoding a transcription activator called EGR-1. This gene is subject to both repression and activation. Binding of WT1 represses transcription of the EGR-1 gene, but without functional WT1 to repress EGR-1, the developing kidney undergoes tumor formation.
Describe splicing of pre-mRNA
Pre-mRNA splicing occurs in enkaryotes. Step 1: Primary transcript combines with suRNPs antl other proleins to form spliceosome. Step 2: Lariat-shaped (looped) inlermetlialc is generated. Step 3: Lariat is released to remove intron precisely and join 2 exons. Patients with lupus make antibodies to spliceosomal snRNPs.
What are mechanisms for transcriptional repression?
Recruitment of chromatin remodeling complexes (opposite of switch sniffs) condense the chromatin Recruitment of histone deacetylases (HDACs) (opposite of HAT) condense the chromatin Recruitment of histone methyl transferase (HMT) proteins bind to methylated histones that silences the chromatin (common method)
What are homeobox domains?
Related to the helix-turn-helix motif. First characterized in several proteins coded by genes concerned with developmental regulation in Drosophila. Homeobox domains contain a helix 3 region that contacts the major groove of DNA and the amino terminus contacts the minor groove.
What is the overall sequential process of transcriptional activation?
Signal Transduction --- Gene Activator Protein Binds to Chromatin --- (chromatin remodeling complex added) --- Chromatin Remodeling --- (histone modification enzymes added) --- Covalent Histone modification --- (other activator proteins added) --- Additional activator proteins bound to gene regulatory region --- (general tx factors, RNA Pol) --- Assembly of pre-initiation complex at promoter --- (other activator proteins, rearrangement of complex) --- Transcription Initiated Positive (activators) and negative (repressors) inputs affect the probability of initiating transcription
What is a silencer?
Sile where negative regulators (repressors) bind Enhancers and silencers may be located close or, far from, or even within (in an intron) the gene whose expression it regulates.
What is the promotor?
Site where RNA polymerase and multiple other transcription factors bind to DNA upstream from gene locus (AT-rich upstream sequence w/ TATA and CAAT boxes). Mutation here commonly results in dramatic drop in amount of gene transcribed. Eukaryotic promoters carry DNA binding elements called cis elements. These sites bind both general and regulatory tx factors (trans factors). Both types of factors help RNA polymerase initiate transcription. The general factors bind to cis elements in the core promoter which is different from the specific regulatory transcription factors (activators) that bind to cis-elements called enhancers. The core promoter is the region where basal factors bind and where RNA polymerase (pol) is recruited. This recruitment of RNA pol is further facilitated by other regulatory sequences outside the promoter that bind sequence specific and nonsequence specific factors of the different classes. Sequences in DNA that bind protein factors are called "cis-elements". The protein factors are called "trans factors".
Describe the steroid/nuclear receptor superfamily as a group of structurally and functionally related transcription factors
Steroid Hormones Diffuse into Cells and Bind to Nuclear Hormone Receptors, which have a DNA Binding Domain and a Hormone Binding Domain. Nuclear hormone receptors then bind to DNA and recruit coactivator proteins. All the ligands are hydrophobic Hormone ligands all share lipophilic properties.
What is the enhancer?
Stretch of DNA that alters gene expression by binding transcription factors. Enhancers and silencers may be located close or, far from, or even within (in an intron) the gene whose expression it regulates.
What is epigenetics?
Study of heritable genetic information that does not involve changes in the nucleotide sequence of DNA, but is manifested as chemical "modifications" that are added to either DNA or chromatin proteins. Ex: DNA methylation and histone modifications such as acetylation and methylation (causing chromatin remodeling).
What transcription factors does RNA Pol II require?
TFII = help to position the RNA Pol (recruited in by mediator) correctly at the promoter. TFIID = its TATA binding protein (TBP) subunit is what recognizes the TATA (cis) element. TFIIH contains helicase activity to facilitate separation of the template strands along with TBP binding. TBP and TFIIH facilitate strand separation in order to transition to the open complex to begin transcription. The RNA pol C-terminal tail extends out of the complex and is a critical target for enzymes that regulate the activity of RNA polymerase. "Activation" of RNA polymerase II requires Phosphorylation (by TFIIH) of the C-terminal tail that extends out of the large RNA pol complex.
What is the difference between hnRNA and mRNA?
The initial transcript is called heterogeneous nuclear RNA (hnRNA) The capped and tailed transcript is called mRNA.
What is the role of chromatin remodeling?
To make DNA less compact
Describe chromatin remodeling via DNA methylation
Transcriptionally active (less compact) chromatin regions tend to be hyperacetylated and hypomethylated. If a region of DNA or a gene is destined for silencing, DNA methyltransferase catalyzes DNA methylation. The methylated DNA has a higher affinity for methyl-CpG binding proteins (like MeCP2) which then recruits histone deacetylases. The net result is similar to repressor proteins that recruit HDACs and histone methyl transferases to repress genes via chromatin compaction Theme -- less access (DNA methylation, histone acetylation) = silencing
What are the general types of DNA motifs?
Zinc finger Helix-turn-helix Leucine zipper Basic-helix-loop-helix
Describe the zinc finger motif?
a DNA-binding domain. There are two major types, the C2H2 and the C4 finger. In the C2H2 finger, the sequence binds one Zn2+ ion through two cysteine (C) and two histidine (H) side chains. In the C4 finger, four critical cysteines in this region each bind one Zn2+ ion. The C4 finger is a distinct form of the motif that is found in the steroid and nuclear receptor superfamily.
