Transcription, RNA Processing, and Control of Gene Expression

Question 1

3 ways RNA differs from DNA

Answer 1

1. Contains ribose instead of deoxyribose
2. Contains uracil instead of thymine
3. Normally single-stranded

Question 2

Significance of DNA template in transcription

Answer 2

RNA is synthesized according to it

Question 3

2 important differences between DNA polymerases and RNA polymerases

Answer 3

1. RNA synthesis can be initiated by RNA polymerase without a primer
2. RNA primases are more error prone (~1/10^9 in DNA, ~1/10,000 in RNA) because RNA does not live very long

Question 4

What class of RNA is ultimately translated into proteins?

Answer 4

Messenger RNA (mRNA)

Question 5

Messenger RNA (mRNA)

Answer 5

Translated into protein, synthesized by RNA polymerase II (Pol II)

Question 6

Ribosomal RNA (rRNA)

Answer 6

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

Question 7

Transfer RNA (tRNA)

Answer 7

Delivers amino acids to ribosome during protein synthesis; synthesized by Pol III; multiple genes for each tRNA found; trimmed and modified extensively

Question 8

What name is given to the DNA sequences that specify the transcriptional start point?

Answer 8

Promoter

Question 9

What name is given to the DNA sequences that specify the transcriptional end point?

Answer 9

Terminator

Question 10

Describe the typical layout of a gene transcribed by RNA polymerase II.

Answer 10

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

Question 11

TFIID (transcription factor for Pol II)

Answer 11

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

Question 12

DNA helicase activity

Answer 12

Separates strands of DNA helix at A/T base pairs to begin transcription

Question 13

By what mechanism does the toxin α-amanitin function?

Answer 13

Inhibits Pol II and causes massive liver failure (essential liver proteins cannot be synthesized); very often fatal (comes from death cap mushroom)

Question 14

How can rifampicin kill certain bacteria yet have low toxicity towards human cells?

Answer 14

Specific and potent inhibitor of RNA polymerase in most bacteria but human Pol II is NOT sensitive to it

Question 15

Capping of pre-mRNA

Answer 15

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

Question 16

Distinguish exons from introns.

Answer 16

Exons are short regions that encode protein that are kept; introns are long regions of non-coding sequence that are spliced out before translation

Question 17

Pre-mRNA splicing

Answer 17

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

Question 18

Specific nucleotide sequences for splicing

Answer 18

• 5’ splice site
• 3’ splice site
• Branch point within intron

Question 19

Outline process of splicing.

Answer 19

1. Adenine residue in branch point of intron attacks 5’ splice site, cutting sugar-phosphate backbone of RNA
2. Cut 5’ end becomes covalently attached to adenine to form a loop in RNA
3. 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)

Question 20

Differential/alternative splicing and its consequences

Answer 20

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

Question 21

Polyadenylation

Answer 21

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

Question 22

What types of RNA are polyadenylated?

Answer 22

Only mRNA

Question 23

Describe how errors in splicing can lead to β-thalassemia.

Answer 23

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

Question 24

Describe how errors in splicing can lead to phenylketonuria.

Answer 24

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

Question 25

Role of chromatin remodeling complexes in availability for gene transcription

Answer 25

Large, multisubunit structures that use ATP hydrolysis to change nucleosome structure temporarily, either unpacking or repacking DNA to allow or repress transcription

Question 26

Role of histone acetyltransferases (HATs) in availability for gene transcription

Answer 26

Acetylation of lysine residues on histones reduces net positive charge of protein, weakening histone/DNA interaction and loosening up structure to facilitate transcription

Question 27

Role of histone deacetylases (HDACs) in availability for gene transcription

Answer 27

Remove acetyl groups from histone to inhibit transcription

Question 28

Role of DNA methylation in availability for gene transcription

Answer 28

Methylation occurs on cytosine, acting as a binding site for particular proteins that recruits HDACs; promotes DNA condensation and inhibits transcription

Question 29

3 classes of DNA binding protein

Answer 29

1. Helix-turn-helix proteins 2. Zinc finger proteins 3. Leucine zipper proteins

Question 30

Key structural features of helix-turn-helix proteins

Answer 30

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

Question 31

Key structural features of zinc finger proteins

Answer 31

Contain zinc as structural element, α-helix involved in DNA binding

Question 32

Key structural features of leucine zipper proteins

Answer 32

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

Question 33

How do the DNA binding proteins recognize specific DNA sequences?

Answer 33

Surface of the protein is complimentary to the special surface features of the double helix in that region; typically bind in the major groove

Question 34

How do DNA binding proteins regulate transcription?

Answer 34

Often recruit proteins with HAT activity

Question 35

LDL receptor

Answer 35

Integral membrane protein that recognizes apolipoproteins B and E of LDL and VLDL

Question 36

How is transcription of the LDL-receptor gene enhanced in response to low cellular cholesterol?

Answer 36

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

Question 37

Hydrophobic hormones

Answer 37

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

Question 38

Cortisol

Answer 38

Hormone secreted by adrenal cortex to stimulate gluconeogenesis, glycogen deposition, and fat and protein degradation, as well as inhibit inflammatory response

Question 39

How does cortisol regulate transcription?

Answer 39

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

Question 40

How do the thyroid hormone receptor and retinoid X receptor (RXR) act together to regulate transcription in response to thyroid hormone?

Answer 40

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)

Question 41

Roles of CREB and CBP in protein kinase A (PKA)-mediated transcription control

Answer 41

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

Question 42

PKA transcriptional control

Answer 42

cAMP --> PKA --> phosphorylated CREB --> binds to promoter elements CREs -> recruits CREB-binding protein (CBP) --> recruits EP300

Question 43

Rubenstein-Taybi syndrome

Answer 43

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

Question 44

What components of signaling network may be defective in Rubenstein-Taybi syndrome?

Answer 44

CBP or EP300

Question 45

Tamoxifen

Answer 45

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