Transcription, RNA Processing, and Control of Gene Expression Flashcards
3 ways RNA differs from DNA
- Contains ribose instead of deoxyribose
- Contains uracil instead of thymine
- Normally single-stranded
Significance of DNA template in transcription
RNA is synthesized according to it
2 important differences between DNA polymerases and RNA polymerases
- RNA synthesis can be initiated by RNA polymerase without a primer
- RNA primases are more error prone (~1/10^9 in DNA, ~1/10,000 in RNA) because RNA does not live very long
What class of RNA is ultimately translated into proteins?
Messenger RNA (mRNA)
Messenger RNA (mRNA)
Translated into protein, synthesized by RNA polymerase II (Pol II)
Ribosomal RNA (rRNA)
Components of ribosome that are required for protein synthesis; 28S, 18S, 5.8S synthesized by RNA polymerase I (Pol I), and 5S is synthesized by RNA polymerase III (Pol III); makes up ~80% of the RNA found in rapidly-dividing cells; multiple copies of each gene are required to allow rRNA to be made to support ribosome synthesis
Transfer RNA (tRNA)
Delivers amino acids to ribosome during protein synthesis; synthesized by Pol III; multiple genes for each tRNA found; trimmed and modified extensively
What name is given to the DNA sequences that specify the transcriptional start point?
Promoter
What name is given to the DNA sequences that specify the transcriptional end point?
Terminator
Describe the typical layout of a gene transcribed by RNA polymerase II.
Can include enhancer (not necessary and can be anywhere before transcription start point), gene specific elements (DNA sequences unique to particular genes), CAAT box/GC rich areas (not always present), TATA box (almost always found); NEEDS GENERAL TRANSCRIPTION FACTORS TO INITIATE TRANSCRIPTION
TFIID (transcription factor for Pol II)
Complex of several individual proteins that binds to TATA box to distort the structure, acting as “signpost” for other transcription factors and resulting in formation of transcriptional initiation complex
DNA helicase activity
Separates strands of DNA helix at A/T base pairs to begin transcription
By what mechanism does the toxin α-amanitin function?
Inhibits Pol II and causes massive liver failure (essential liver proteins cannot be synthesized); very often fatal (comes from death cap mushroom)
How can rifampicin kill certain bacteria yet have low toxicity towards human cells?
Specific and potent inhibitor of RNA polymerase in most bacteria but human Pol II is NOT sensitive to it
Capping of pre-mRNA
Cap containing methylguanosine is added to 5’ end of pre-mRNA, distinguishing it from other types of RNA (only mRNA gets it); important for protection against degradation and initiation of translation
Distinguish exons from introns.
Exons are short regions that encode protein that are kept; introns are long regions of non-coding sequence that are spliced out before translation
Pre-mRNA splicing
Catalyzed by spliceosomes (referred to as snRNAs), complexes which recognize boundaries between exons and introns; introns are removed and exons joined together to make mature mRNA; involve protein and snRNA components
Specific nucleotide sequences for splicing
- 5’ splice site
- 3’ splice site
- Branch point within intron
Outline process of splicing.
- Adenine residue in branch point of intron attacks 5’ splice site, cutting sugar-phosphate backbone of RNA
- Cut 5’ end becomes covalently attached to adenine to form a loop in RNA
- Newly generated 3’ end of exon reacts with 5’ end of next exon to join exon sequences and release intron as lariat (loop with covalent bond at adenine)
Differential/alternative splicing and its consequences
Splicing allows more than one protein product from single pre-mRNA transcript from single gene (ex. tropomyosin gene can form striated muscle, smooth muscle, myoblasts, non-muscle fibroblasts, and brain tissue from different splicing patterns); increases repertoire of proteins and allows certain splicing patterns to form type-specific isozymes
Polyadenylation
Polyadenylation signal encoded by genome is recognized by specific protein factors and cleavage of RNA molecule is catalyzed, causing a new 3’ end to form; poly-A polymerase adds hundreds of adenine residues (forming the poly A tail) to 3’ end, giving stability to mRNA
What types of RNA are polyadenylated?
Only mRNA
Describe how errors in splicing can lead to β-thalassemia.
β-thalassemia is imbalance of β-globin gene synthesis leading to lack of hemoglobin; mutation causes splice site to be mutated so that two mutant mRNA are produced
Describe how errors in splicing can lead to phenylketonuria.
Phenylketonuria is often due to mutation at 5’ splice donor site that leads to a protein with a missing exon that gets rapidly degraded, which causes lack of enzyme activity so that PKU symptoms occur
Role of chromatin remodeling complexes in availability for gene transcription
Large, multisubunit structures that use ATP hydrolysis to change nucleosome structure temporarily, either unpacking or repacking DNA to allow or repress transcription
Role of histone acetyltransferases (HATs) in availability for gene transcription
Acetylation of lysine residues on histones reduces net positive charge of protein, weakening histone/DNA interaction and loosening up structure to facilitate transcription
Role of histone deacetylases (HDACs) in availability for gene transcription
Remove acetyl groups from histone to inhibit transcription
Role of DNA methylation in availability for gene transcription
Methylation occurs on cytosine, acting as a binding site for particular proteins that recruits HDACs; promotes DNA condensation and inhibits transcription
3 classes of DNA binding protein
- Helix-turn-helix proteins
- Zinc finger proteins
- Leucine zipper proteins
Key structural features of helix-turn-helix proteins
Contains two α-helices connected by short chain of amino acids; R groups/side chains of amino acids in one helix is involved in DNA recognition
Key structural features of zinc finger proteins
Contain zinc as structural element, α-helix involved in DNA binding
Key structural features of leucine zipper proteins
Dimers where α-helix from each monomer forms coiled coil structure, typically containing leucine at every seventh position to line up on one face; interact with basic region on DNA
How do the DNA binding proteins recognize specific DNA sequences?
Surface of the protein is complimentary to the special surface features of the double helix in that region; typically bind in the major groove
How do DNA binding proteins regulate transcription?
Often recruit proteins with HAT activity
LDL receptor
Integral membrane protein that recognizes apolipoproteins B and E of LDL and VLDL
How is transcription of the LDL-receptor gene enhanced in response to low cellular cholesterol?
Transcription of LDL receptor gene is upregulated in response to low cellular cholesterol to result in more LDL receptor production and enhanced cholesterol uptake from blood; requires basal factors SP-1, CRSP, and general transcription factors, but also SREBP-1a, which is triggered by low cellular cholesterol – binds to SRE and recruits HAT and other proteins to cause loosening of the DNA to enhance transcription
Hydrophobic hormones
Cross plasma membrane of cells to bind to soluble receptors in cytoplasm/nucleus; hormone/receptor complex binds to DNA at specific binding sites to regulate transcription
Cortisol
Hormone secreted by adrenal cortex to stimulate gluconeogenesis, glycogen deposition, and fat and protein degradation, as well as inhibit inflammatory response
How does cortisol regulate transcription?
Ex. Phosphoenolpyruvate carboxykinase (PEPCK) is regulated by cortisol binding and activating receptor in cytoplasm, causing conformational change, nuclear import, binding to gene, and recruitment of additional proteins to enhance transcription levels
How do the thyroid hormone receptor and retinoid X receptor (RXR) act together to regulate transcription in response to thyroid hormone?
In the absence of bound thyroid hormone, they form a dimer to recruit HDAC activity, maintaining chromatin in a condensed state and preventing transcription (when thyroid hormone is present, transcription is activated by HAT)
Roles of CREB and CBP in protein kinase A (PKA)-mediated transcription control
CREB is phosphorylated by PKA, and then is bound to CRE to recruit other proteins such as CREB binding protein (CBP), recruiting EP300 and then interacting with general transcription factors to function as HATs
PKA transcriptional control
cAMP –> PKA –> phosphorylated CREB –> binds to promoter elements CREs -> recruits CREB-binding protein (CBP) –> recruits EP300
Rubenstein-Taybi syndrome
Inherited condition causing particular facial features, intellectual disability, broad thumbs and first toes, and growth retardation; caused by mutations in gene encoding CREB binding protein (CBP), which affects CREB-mediated transcription processes which are important in many things, including memory and cognition
What components of signaling network may be defective in Rubenstein-Taybi syndrome?
CBP or EP300
Tamoxifen
Hormone drug that inhibits estrogen action by binding to estrogen receptor as a competitive inhibitor, blocking estrogen binding; treatment for breast cancer by disrupting proliferation of cells in the breast; often resistance occurs
