Detail three important ways in which RNA differs from DNA
- Contains ribose rather than deoxyribose as sugar - Contains four bases adenine, guanine, cytosine and uracil - Single-stranded structure
List two important differences bw DNA polymerases and RNA polymerases
- RNA polymerase can initiate synthesis without a primer - RNA polymerase is more error prone than DNA polymerase, no proofreading activity
In what direction does RNA pol synthesize RNA?
- 5 to 3’
What class of RNA is ultimately translated into proteins?
What names are given to the DNA sequences that i.) specify the transcriptional start point and ii.) specify the transcriptional end point?
i.) Promoter ii.) Terminator
Describe the typical layout of a gene that is transcribed by RNA polymerase II
- Enhancer (way upstream) - Gene-specific elements: DNA sequences unique to particular genes or groups of genes - CAAT box or GC-rich region: may be present - TATA box: almost invariably found in genes transcribed by Pol II - Gene
What is the name of the molecule that synthesizes mRNA?
- RNA pol II
What is the name of the molecule that synthesizes rRNA?
- RNA pol I, some Pol III
What is the name of the molecule that synthesizes tRNA?
- RNA Pol III
Can RNA Pol II initiate transcription of DNA? Explain
- No it cannot, it requires help of GTFs
What GTFs are required by RNA Pol II?
- TFIID binds to TATA box, which distorts the DNA helix, acts as signpost and recruits remainder of factors and pol II - Transcriptional initiation complex forms
What sequence are promoter regions rich in? Why?
- Rich in AT sequences and presence of TATA box. - Easier to separate AT base pairs than GC base pairs, by DNA helicase activity of one of the GTFs
What is the basal transcription complex?
- RNA pol II with GTFs = basal transcription complex - This initiates transcription at a low rate
Discuss rate of DNA transcription in context of basal transcription complex
- Basal transcription complex = RNA pol II with GTFs - This complex initiates transcription at low rates - High rate requires NF1 binding to CAAT box and SP-1 binding to GC-rich sequences
By what mechanism does the toxin alpha-amantin function? How are ppl exposed to this? Symptoms? Treatment?
- Death cap mushroom common in cool, damp regions of pacific coast - Genus/species: amanita phalloides - Contains alpha-amantin, which is a Pol II inhibitor - Nausea, vomiting, coma, hepatic encephalopathy from liver failure - Liver failure ~ 48 hour post-ingestion - Tx: gastric lavage, activated charcoal and liver transplant
How can rifampicin kill certain bacteria yet have low toxicity towards human cells?
- Used to treat mycobacterial infections such as TB and leprosy - It is a specific inhibitor of RNA pol found in many types of bacteria, but euk Pol II is not sensitive to it
3 mRNA processing steps
- Capping - Splicing - Polyadenylation
What is meant by the capping of pre-mRNA? Is the cap unique to pre-mRNA? Function?
- Methylguanosine is added to the 5’ end of pre-RNA - This distinguishes mRNA from other types of RNA - Required for subsequent processing steps - Protects against degradation - Important for initiation of translation
Distinguish exons from introns
- Exons: short regions encoding protein - Introns: long regions of non-coding sequence
Discuss pre-mRNA splicing.
- Catalyzed by complex known as spliceosome, which has protein and snRNA compoments involved in catalysis and recognizing exon/intron boundaries - Consensus nt seqs are required for splicing: 5’ splice site, 3’ splice site and branch point within intron - Adenine at branch point attacks 5’ splice site, cutting sugar-phosphate backbone of RNA - 5’ end becomes linked to adenine, forms a loop - Free 3’OH reacts with 5’ end of next exon, joins. Intron released as lariat
What is meant by differential or alternative splicing? What are the consequences of this process?
- Splicing allows for more than one protein product to be generated from a single pre-mRNA transcript, which is called differential/alternative splicing - Increases repertoire of proteins produced – ie. catalytic properties, subcellular locations, distinct interactions differ.
What is polyadenylation? Describe. What types of RNA molecule are polyadenylated?
- Polyadenylation = addition of 100s of adenine residues to end of mRNA moledule - Stop pt for transcription is specified by a consensus seq in DNA known as polyadenylation signal, which is transcribed into pre-mRNA - This signal is recognized by specific protein factors - This tail is important for RNA stability
Where does RNA processing occur? Does pre-mRNA leave the nucleus?
- Nucleus - No, only mature mRNA
Describe mRNA stability
- mRNA molecules decay under ribonucleases - There half lifes are variable from hours to minutes
- Reduced beta-chain of hemoglobin due to mutation in beta-globin gene at first exon/intron boundary. Mutation at 5’ splice site causes two aberrant mRNA products to be formed
Describe molecule basis for PKU
- Inability to convert phenylalanine to tyrosine due to mutation often at 5’ splice donor site in phenylalanine hydroxylase gene - Results in defective splicing, protein produced lacks one exon and is rapidly degraded
What roles do chromatin remodelling complexes, histone acetyltransferases (HATs), histone deacetylases (HDACs) and DNA methylation play in controlling the availability of genes for transcription?
1.) chromatin remodeling complexes: use ATP to change nucleosome structure temporarily to allow or inhibit transcription 2.) HATs: acetylate lysine residues in histones, reduce net pos charge of protein, weakens histone:DNA interaction, facilitates transcription 3.) HDACs: remove acetyl groups from histone, increasing net pos charge of protein, strengthens histone:DNA interaction, inhibits transcription 4.) DNA methylation: methyl groups added to cytosine, found in silent regions of genome, binds proteins that recruit HDACs to DNA, promoting condensation and inhibiting transcription
Describe key structural features of three classes of DNA-binding protein, namely helix-turn-helix proteins, zinc finger proteins and leucine zipper proteins.
1.) Helix-turn-helix proteins: two alpha-helices connected by short AA chain. Side chains of AA in one helix recognize DNA 2.) Zinc finger proteins: zinc as structural element, alpha-helix binds DNA 3.) Leucine zipper proteins: dimers where alpha-helix from each monomer forms coil structure, leucine typically found at every seventh position in sequence of polypeptide chain forming alpha-helix
In general, how do DNA-binding proteins regulate transcription?
- Binding of these proteins recruit other proteins to DNA, including HATs, chromatin remodeling complexes or HDACs
Explain how LDL receptor production changes in response to cholesterol. Discuss mechanism
- Transcription of LDL receptor gene is upregulated in response to low cellular cholesterol - Result: more LDL receptor production and enhanced cholesterol uptake from blood Low-level transcription - SP1 (zinc-finger protein) binds to GC rich regions in LDL receptor promoter - SP1 requires CRSP protein cofactor to be activated - SP1 and CRSP help pol II and GTFS to assemble at promoter - These are all considered basal transcription factors Up-regulated transcription - Requires basal transcription factors - Also SREBP-1a enters into nucleus as triggered by low cellular cholesterol - SREBP-1a binds to SRE and recruits HAT activity plus some other proteins - Result = enhanced transcription of LDL-receptor gene
How does the steroid hormone cortisol regulate transcription?
- Cortisol is secreted by adrenal cortex - Binds to and activates receptors in cytoplasm, causing conformational change to that receptor - Accessory proteins dissociate, activated receptors dimerize - Dimerized receptors are imported into nucleus - Bind GRE elements on genome, regulate transcription positively or negatively - Typical result of cortisol is to stimulate gluconeogenesis, glycogen deposition, fat and protein degradation, inhibit inflammatory response
Describe regulation of transcription in response to thyroid hormone
- Thyroid hormone becomes bound to receptors that are constitutively in the nucleus and are bound to DNA elements of genes that respond to it - THR (receptor) when not bound to thyroid hormone is dimerized with RXR (retinoid X receptor) - This dimer recruits HDAC, which condenses chromatin and inhibits transcription - Binding of thyroid hormone triggers conformation change in THR, HDAC is released and HAT binds in its place, leads to relaxation of chromatin and enhancement of transcription - Response: stimulated metabolic rate in most tissues
What is Rubinstein-Taybi syndrome?
- Inherited condition characterized by hypoplastic maxillar (insufficient growth of midface), prominent nose, intellectual disability, polydactyly, growth retardation - Results from mutation in gene encoding CREB binding protein (CBP) - This gene is implicated in processes related to memory, cognition and developmental processes
Discuss roles of the CREB, CBP and PKA in transcriptional control
- PKA phosphorylates and regulates many cytosolic proteins, but is also present in activated form in nucleus under high levels of cAMP - Many genes regulated by PKA contain CRE (cAMP responsive element) in their promoter region - PKA influences transcription by phosphorylation of CREB, which then binds CRE - CREB bound to CRE recruits CBP - CBP then recruits EP300 - CBP/EP300 interacts physically with GTFs. Together CBP/EP300 function as HATs, relaxing chromatin and stimulating transcription
What is the mechanism of action of tamoxifen?
- Tamoxifen inhibits estrogen action - It is a competitive inhibitor, blocking estrogen binding to its receptor, however ERE dimer still remains dimerized and bound to DNA - Growth of breast cancer is reduced by disrupting this estrogen signaling