UPenn MSGC advanced exam 2

The sense strand of RNA is equivalent to the

coding strand of DNA

The antisense strand of DNA is equivalent to the

template strand of DNA

core promoter elements in bacteria

TATA box (-10) and TTGACA (-35)

which RNA pol produces mRNA?

RNA pol II

which RNA pol produces almost all rRNA?

RNA pol I

which RNA produces tRNA and 5s rRNA?

RNA pol III

additional core promoter elements - not present in all promoters

BRE (B recognition element)

DPE (downstream promoter element)

INR (initiator element)

general transcription factors

TFIID

TFIIB

TFIIA

TFIIF

TFIIE

TFIIH

TFIID

general transcription factor that recognizes the TATA box

composed of TBP and other TAFs

TFIIH

helicase for transcription initiation

phosphorylates RNA pol CTD to release it from the promoter

two functional domains in TFs

DNA binding domain

trans-activating domain

which TF initiates transcription?

TFIID - binds promoter at TATA box with the TBP subunit

transition from initiation to elongation

TFIIH phosphorylates the C-terminal domain of RNAP II to release it from the other TFs and begin elongation

eukaryotic transcription also requires

transcriptional activators, a mediator protein, chromatin remodeling enzymes

what three enzymes are involved in mRNA capping?

phosphatase

guanyl transferase

methyl transferase

phosphatase role in capping

removes one phosphate group from the 5’ end of the growing chain

guanyl transferase role in capping

transfers a GMP through a 5’-5’ linkage

methyl transferase role in capping

adds a methyl group to the 7 position of guanosine 5’ end creating a 7-methylguanosine cap

Cap binding complex (CBC)

proteins that remain bound to the cap until translation initiation

assists in processing and export of mature mRNA

which genes lack introns

histones

interferons

sequence elements needed for intron excision

5’ end: exon ends with MAG and intron begins with GURAG

3’end: intron ends with CAG and exon begins with G, polypyrimidine tract upstream of this

lariat formation key factors

adenine - in intron, attacks the 5’ splice site in the intron to cut the backbone

3’OH at the end of the exon reacts with the start of the next exon to release the intron

RNA elements of the spliceosome

U1, U2, U4, U5, U6 - enzymatic catalysis

U3 is not involved in splicing but is another snRNA

splicing snRNA & protein steps

1. U1 base pairs with 5’ splice junction

2. BBP reocgnizes branch point site and binds with U2AF

3. U2 base pairs with branch point site, displaces BBP and U2AF

4. U4/U6-U5 triple snRNP enters and displaces U1 - creates active site for splicing

5. U2 and U6 RNAs bring the intron/exon junctions together to form active site

6. lariat is formed and two exon segments come together in the active site

7. EJC mars splicing as complete

8. intron released as lariat

SLO and alternative splicing

SLO encodes a calcium-regulated potassium channel. One splice variant creates a protein with low calcium sensitivity, another creates a protein with high calcium sensitivity

3’ end formation key elements

AAUAAA and GU/U rich element

polyA polymerase (PAP)

cleavage stimulation factor (CstF)

polyadenylation specificity factor (CPSF)

polyA binding protein (PAB) - immediately binds the polyA tail

eukaryotic transcription termination mechanism

RNAPII continues transcription after transcript made, but transcript formed downstream of cleavage site lacks cap

lack of cap results in rapid degradation and signals RNAPII to stop

FSHD inheritance

AD

FSHD prevalence

about 1:8,000 - 1:38,000

among the most common myopathies affecting global populations

what makes FSHD different from most muscular dystrophies?

symptoms present in adulthood rather than childhood

FSHD primary muscle groups affected

face, scapular, and humeral muscles (axial and leg)

FSHD variability & penetrance

highly variable, reduced penetrance (30% of family members with variant will be non-penetrant or minimally affected)

FSHD phenotype

slow but variable progression, periods of stability and rapid deterioration, scapular winging, 20% of patients need wheelchairs after 50, often asymmetric weakness

age of onset typically between 15-30

FSHD genetic cause

4q35

regions of repeats in DUX4 contracted

leads to de-repression in skeletal muscle (loosened chromatin)

4qA causes stable DUX4 transcript

repeats in FSHD - affected and population

normal D4Z4: 8-100 repeats

FSHD D4Z4: 1-10 repeats

FSHD 4qA and 4qB mechanism

FSHD is only possible with contractions of DUX4 gene

if contractions, pt needs 4qA at the distal end of the gene to be affected

4qB pts with contractions are still unaffected because the transcript is unstable and cannot be translated

FSHD1 vs FSHD2

FSHD1 is more common, 1-10 repeats

FSHD2 is less common (5% of patients), 9-20 repeats, mutation in genes that affect chromatin density causes the expression of DUX4 gene

Mutated genes in FSHD2 - locus heterogeneity

SMCHD1 (85%) DNMT3B (rare)

mechanism of the protein in FSHD

toxic gain of function in DUX4 gene - TF not normally expressed in adult cells, triggers cytotoxic cascades that cause apoptosis of muscle cells

mRNA “export ready” signals for nuclear pore recognition

cap binding complex

exon junction complex

polyA binding proteins

RNA vaccine technology

antigen-encoding mRNA transported into a cell and translated into antigen protein

elicits humoral and cellular immunity by presenting mRNA-encoded antigen

mRNAs are contained within lipid nanoparticles

can be used in cancer cells, but very expensive

nonpolar/hydrophobic amino acids

Alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, tryptophan

proline important distinction

causes a mend in the amino acid chain

branched chain amino acids

valine, leucine, isoleucine

cause MSUD, proprionic acidemia, and isovaleric acidemia when defected

hydrophilic amino acid facts to know

glycine is the smallest

serine, threonine, and tyrosine all have hydroxyl group available for phosphorylation

ser/thr kinases make up many regulatory proteins

cysteine important distinction

ends with a sulfhydryl group (-SH) that can form covalent links with other sulfhydryls

polar & basic amino acids

lysine, arginine, histidine

polar and acidic amino acids

aspartic acid, glutamic acid

features of the genetic code

the genetic code is unambiguous, degenerate, comma-less, non-overlapping, and nearly universal

aminoacyl-tRNA synthetase

add an amino acid to the 3’ end of a tRNA molecule

only 20 (one per amino acid)

tRNA activation - 2 steps

amino acids bound at the carboxylic acid group

1. amino acid activated by attachment of carboxyl group to AMP

   1. amino acid transferred to the 3’OH of the tRNA

health conditions associated with tRNA synthetases

neuropathy (CMT), deafness: lysine & glycine

fatal infantile hypertrophic mitochondrial cardiomyopathy: alanine

growth retardation, ID, hypotonia, hepatopathy: isoleucine

combined oxidative phosphorylation deficiency: threonine

deafness, female infertility: histidine

polymyositis/dermatositis - autoimmune disorder associated with arthritis, myositis, interstitial lung disease, Raynauds: antibodies found against one or more of the tRNA synthetases

eukaryotic ribosome subunits

60S - rRNAs = 28S, 5.8S, and 5S, plus 49 proteins

40S - small subunit

where is most rDNA found?

the p arms of the acrocentric chromosomes (13, 14, 15, 21, 22)

45S RNA

the large precursor for all rRNA except 5S

5S rDNA details

located on chromosome 1 and synthesized by RNA pol III

translation initiation requires

small and large ribosomal subunits

GTP

a specific charged tRNA (initiator tRNA)
Mg2+

translation initiation factors

Kozak sequence

the specific sequence in eukaryotic mRNA that helps identify the correct AUG to start on

5’-ACCAUGG-3’

which is the only tRNA that can bind the small ribosomal subunit without the large one present?

initiator tRNA (distinct from tRNA-Met)

translation initiation steps

1. small ribosomal subunit loaded with initiator tRNA-Met, GTP and initiation factors (eIFs)

2. this complex recognizes the 5’cap and binds mRNA, 2 eIFs already bound; at the same time, PABP is contacted to ensure that the mRNA has a polyA tail

3. complex scans the mRNA looking for the Kozak sequence; at same time, additional eIFs act as helicases to disrupt mRNA secondary structure

   1. eIFs dissociate from complex at the AUG site & large ribosomal subunit binds

where is the tRNA-met bound after initiation

the P site

which terminal of the polypeptide chain are amino acids added to?

C-terminal end

translation elongation steps

1. incoming charged tRNA loads onto the ribosome at the A site bound by EF-1 and GTP

2. GTP is hydrolyzed

3. peptidyl transferase forms the peptide bond via RNA catalysis

4. the uncharged tRNA moves to the E site & the second tRNA with the polypeptide chain attached moves to the P site using EF-G and GTP hydrolysis

5. additional amino acids added in the same fashion until a stop codon is reached

translation termination steps

1. stop codon in the A site (UAG, UAA, UGA)

2. GTP-dependent release factors bind - eRF1 recognizes the stop codons and eRF3 is the GTPase that helps in release

3. peptidyl transferase transfers a water molecule to the amino acid chain instead of an amino acid

   1. peptide chain released, mRNA released, ribosome dissociates into subunits

IRES - internal ribosome entry site

allows for an alternative mechanism of translation initiation to occur when typical translation is impaired (cell stress or viral infection)

typically in the 5’ UTR

has a specific structure to initiate translation without a cap

ITAFs

IRES trans-acting factors

regulate IRES translation

problems with IRESs and ITAFs

can contribute to tumorigenesis and resistance to therapies

dysregulation is related to neuro and cardio diseases, muscular dystrophym and other syndromes

RAN translation

repeat-associated non-AUG translation

repeated sequences create a secondary structure in mRNA, which facilitates translation that begins at a non-AUG start close to the repeat

produces small peptides of repeated amino acids based on the repeat sequence

identified in nucleotide repeat disorders research

RAN translation in fragile X

CGG repeats found in the 5’ UTR of the FMR1 gene (upstream of typical AUG start site)

RAN initiated translation produces toxic homopolymer proteins that contribute to neurodegeneration in Fragile-X associated tremor/ataxia syndrome (FMRpoly(R), FMRpoly(G), FMRpoly(A))

alpha helix secondary structure details

stabilized by H bonds between NH and CO groups in the peptide backbone

coil causes amino acids 3-4 apart in the linear sequence to be adjacent in the helix

protein domains

smaller parts of a protein that usually fold independently into structural units and typically have a specific function

what type of protein structure is at risk to poisoning?

quaternary proteins - can have dominant-negative mutations

p53 - a tetramer protein - mutation types

dominant-negative - mutant subunit binds with wildtype subunit and interferes with function

LOF - no protein or no function due to mutations

GOF - mutation turns on function when it’s not supposed to be on

what s the largest known human gene and how many exons does it have?

dystrophin, 79 exons

DMD vs BMD

DMD is more severe, onset is younger (2-5 vs 15 years), rapid progression vs slow progression, life expectancy in DMD is late teens or early 20s, in BMD is 40-50 years

DMD/BMD inheritance

X-linked

DMD/BMD general symptoms

progressive muscle weakness, scoliosis, cardiomyopathy

without family history, a woman with a child with DMD has ___ chance of being a carrier ( ___ chance it’s de novo)

2/3 chance of being a carrier, 1/3 chance it’s de novo

Dystrophin protein - functions and consequences of loss

expressed in muscle cells, part of a complex that stabilizes the plasma membrane of striated muscle cells

loss of dystrophin causes instability of the cell membrane and myofiber loss

lost muscle fibers are replaced by fibrosis and fat, causing progressive muscle weakness

dystrophin gene mutation hotspots

exons 45-55 and 3-9

dystrophic deletion/insertion mutations (DMD vs BMD)

DMD - deletion or duplication is out of frame

BMD - deletion or duplication is in frame - still reduces gene function some

FDA approved ASO treatment for DMD

ASO = antisense oligonucleotide

Exondys 51 - targets exon 51 to bring the mutation back into frame

essentially going from DMD to BMD

weekly IV infusion - not the same as gene therapy which is one and done

what is the goal of exondys in DMD progression?

producing more protein to slow the decline, NOT fixing the damage already done

DMD gene therapy

Elevidys - “minigene” in adeno-associated virus provides a functional microdystrophin protein to cells

what type of mutation causes hemoglobin S?

sever missense mutation

hemoglobin S associated condition

Sickle cell anemia

sickle cell anemia mechanism of disease

A>T missense mutation in HBB gene causes RBCs to sickle and clog capillaries

causes pain, organ damage, and increased risk for stroke

Beta-thalassemia mechanism

GOF dominant-negative

mutated protein (stop codon in the last exon) interferes with normal protein causing severe AD anemia

nonsense mediated decay (general)

mechanism that cells use to decrease the production of truncated proteins

nonsense mediated decay mechanism/steps

in typical mRNA, the commonly used stop codon is in the last exon, and all splicing is upstream of that

in a full length protein, all exon junction complexes should be displaced by ribosome during translation, so if there is EJC upstream of the stop codon, the ribosome can sense a problem

after sensing this, proteins associated with the ribosome will initiate a different pathway: cap is removed and mRNA is degraded

this all prevents production of truncated protein

exceptions to nonsense mediated decay

premature stop codon in the last exon - no EJC after it for the ribosome to sense

PTC is <50nts upstream from the last exon junction - too close for the machinery to catch it

in addition to protecting the cell from variants, ______ acts as an anti viral system.

nonsense-mediated decay

how do antibiotics target prokaryotes?

they target protein synthesis in the specific ribosomes that prokaryotes have (which are different in size and shape to eukaryotic ribosomes)

antobiotics that interfere with bacterial protein synthesis

tetracycline, streptomycin, chloramphenicol, ertythromycin

translational readthrough

allows tRNAs to incorrectly pair with stop codons to continue translation

could be beneficial to target nonsense mutations by essentially replacing them with missense mutations

mediated by near-cognate aa-tRNAs, so complementary to 2/3 positions of the stop codon

initiated by aminoglycosides

how do aminoglycosides promote translational readthrough?

alter the ribosome to reduce discrimination between near-cognate aa-tRNAs and release factors

DNA methylation is associated with _____ gene expression

decreased

which DNA base is methylation limited to?

cytosine

in what sequence to methylated C’s occur?

5’-CG-3’ which reads the same on both strands and allows for symmetric methylation

how does methylation impact cytosine?

base pair specificity is unchanged, but the methyl group protrudes into the major groove and can affect protein binding