EXAM I Zaidi Flashcards
Function of mRNA and miRNA
mRNA - RNA molecules copied from genes that direct protein synthesis
Code for proteins
miRNA - MicroRNAs; regulate gene expression by blocking translation of selective mRNA causing their degradation
Function of rRNA and tRNA
rRNA - form the core of ribosome and catalyze protein synthesis
tRNA - adaptors that select and hold aa during protein synthesis
Function of snRNA, snoRNA, siRNA
snRNA - small nuclear; splice pre-mRNA to form mRNA
snoRNA - small nucleolar; process and chemically modify rRNAs
siRNA - small interfering RNA; regulate eukaryotic gene expression by degrading select mRNA
Function of RNA polymerase; name the ones used for prok and euk transcription
Catalyzes the formation of phosphodiester bonds
RNA synthesis has lower fidelity than DNA synthesis due to the mistakes not being transmitted to progeny (RNA is a transient copy)
Prokaryotes - RNA Polymerase
Eukaryotes - RNA Polymerase II (Euks require 3 polymerases)
List the basic steps of transcription
Initiation
Elongation
Termination
Describe initiation of transcription in prokaryotes
(main regulatory step)
RNA pol slides until locates a promoter
sigma subunit binds to promoter = open promoter complex
Short DNA strand is unwound into a transcription bubble
Rxn is driven forward via the hydrolysis of the nt triphosphates (ATP, CTP, UTP, GTP)
Transcription promoters in proks
Transcription promoters in euks
5’ upstream of start site = -35 sequence (TTGACA), -10 sequence (TATAAT)
Euks = TATA box (or CAAT box, GC Box)
Elongation of Prok Transcription; what enzyme releives tension? What causes termination? What is the termination signal?
RNA pol & promoter separate, sigma factor dissociates
DNA gyrase releives superhelical tension
GC then AC rich region = hairpin loop with poly(U) tail at the end
Describe initiation of transcription in eukaryotes, what complex is formed; what initiates elongation?
TFIID binds to TATA box via the TATA-box binding protein (TBP)
The formation of the transcription initiation complex (TFIIB, TFIIF, RNA Pol II, TFIIE, TFIIH)
DNA is unwound exposing template strand
Elongation initiated with polymerase leaves promoter by CTD phosphorylation by TFIIH
What unique sequence does eukaryote polymerase II contain and what regulates it? What are the general TFs?
carboxyl-terminal domain = CTD
Regulated by phosphorylation on Ser residues of the CTD
TFs: TFII - assemble promoter before transcription
positions RNA poly, aids in pulling DNA strands apart, releases RNA rolymerase into elongation mode
Describe transcription elongation in eukaryotes, what prevents the dissociation of RNA pol II? What removes tension?
RNA Pol II moves along DNA transcribing RNA along the way
Elongation factors prevent RNA Pol II dissociation
DNA topoisomerase removes tension
What additional proteins are required during euk transcription which allow for initiation to occur due to it being highly compacted?
- Transcriptional activator - help attract RNA Pol II to transcription initiation start site
- Mediator - allows activator proteins to communicate w/ RNA Pol II and general TFs
- Chromatin modifying enzymes - provide greater access to DNA with the help of chromatin remodeling complexes and histone modifying enzyme
List the 3 Post-transcriptional modifications in eukaryotes
- 5’ RNA cap (7-methyl guanosine) - stabilizes & protects mRNA 5’ from phosphatases & nucleases
- RNA splicing - intron removal - exons exit with final mRNA (DO NOT OCCUR IN BACTERIA!!!)
- Polyadenylation at 3’ end - poly A tail - important proteins Cleavage & Polyadenylation Specificity Factor (CPSF; binds poly A tail), Cleavage Stimulation Factor (CstF; binds GU rich element) RNA is cleaved & RNA polymerase adds 200 nts where the Poly A binding proteins can assemble
Define the adaptor hypothesis of tRNA
The genetic code is read by molecules that can recognize a codon and carry the corresponding amino acid
tRNA is an adaptor that binds to specific codons and brings with it an aa for incorporation into the polypeptide chain
List the 2 important structures of tRNA
Anticodon loop - pairs with complementary codon in an mRNA molecule
3’ CCA terminal region - binds the aa that matches the corresponding codon
Function of aminoacyl-tRNA synthetase
What allows it to be accurate?
Catalyzes the attachment of the correct aa to the corresponding tRNA to the 3’ terminal ribose residue forming the activated aminoacyl-tRNA driven by ATP hydrolysis
Hydrolytic editing - accurate tRNA charging
Which direction is protein synthesized in?
N-terminal to C-terminal
What 4 steps occur during elongation during translation?
tRNA binding
peptide bond formation
large subunit translocation
small subunit translocation
What must occur for initiation during translation?
initiator tRNA carries AUG = Met (methionine)
Initiator tRNA-met complex is loaded onto the small ribosome P site along with other euk initiation factors (eIFs) = eiF4E and eIF4G
Small subunit scans until AUG is reached
Small + Large subunit join = addition of aminoacyl tRNA
What elongation factors are involved during translation in eukaryotes & bacteria?
EF-Tu, EF-G = bacteria; EF-TU binds GTP and aminoacyl tRNA which monitors interaction b/w anticodon and aminoacyl tRNA & codon of mRNA = Proofreading
EF1, EF2 = eukaryotes
What recognizes the stop codon during translation? Where do they bind?
Release Factors (RFs)
Bind to A site, catalyzes the addition of water, freeing the carboxyl group from tRNA
List the 3 major forms of transport in cells; which organelles are involved in each?
Gated - nucleus + cytosol
Transmembrane - from cytosol across an organelle membrane
Vesicular - membrane-enclosed transport intermediates
Features of nuclear transport; through which structure do these proteins move through to exit and enter the nucleus?
What is the nuclear localization signal for import into nucleus? What recognizes these signals?
What is the signal for mitochondrial translocation?
ER Signal sequence?
Gated, bidirectional, selective
Used for proteins that are needed in the nucleus (histones, DNA, RNA polymerase, topoisomerase, TFs, etc.)
Proteins moving into cytosol = tRNA, mRNA
Nuclear Pore Complexes (NPCs) - fibrils facilitate transport with their FG rich binding site for the import receptors
NLS = (+) lysine and arginine via nuclear import receptors
Mitochondrial contain (+) charged residues on one end and hydrophobic on other end
ER - vary in aa sequence, non-polar aa w/ guided by SRP & SRP receptor
What 3 mechanisms involve the controlling of protein transport?
Phosphorylation
Proteolysis
Binding to Inhibitory Proteins
Where are functions TIM 22 and TIM 23 in mitochondria translocation?
SAM and OXA?
TIM 22 - soluble protein —> matrix and helps insert membrane proteins in inner membrane
TIM 23 - subclass entrance into mito (i.e. ATP, ADP, Pi)
SAM - inserts/folds beta-barrel proteins in OM
OXA - protein insertion synthesized in mito
How are unfolded proteins maintained during mitochondria translocation?
What cleaves the signal sequence once inserted?
Hsp 70
Cleaved via peptidase
ER Signal sequence
ER Signal Recognition Particle (SRP)
Signal Sequence: Vary in aa; non-polar aa, large hydrophobic pocket
SRP
What are the 3 vesicular transport systems? BER
Biosynthetic pathway
Endocytic pathway
Retrieval pathway
What are the two functions of the vesicular coats? What are 3 types of coats?
Concentrates select proteins
Molds the vesicle
Coats are usually discarded before vesicle fuses w/ target membrane
COPI, COPII - ER & Golgi transport (COPII FROM ER EXITS SITES)
Clathrin-coated - Golgi & Plasma membrane transport
Role of PI (phosphoinositides) in vesicular trafficking?
Undergo rapid cycles of phosph and dephos at 3’,4’,5’ positions
Interconversion = controls recruitment and binding of proteins to specific organelles/domains and regulate vesicle trafficking
How does the clathrin coat remove from the vesicle? What protein is involved?
Dynamin assembles ring with a PIP2 binding domain and GTPase domain
Pinching
Distortion of lipid bilayer
Hsp70 peels off coat
Function of Rab and SNARE in vesicle targeting?
Rab - direct vesicle to specific spots on target membrane
Involved in specificity; Rab-GDP = inactive, soluble; Rab-GEFs = active GTP; tightly bound to membrane
SNARE - mediates fusion of vesicle with membrane
Rab1
Rab2
Rab3A
1 = ER and Golgi
2 = cis golgi
3 = synaptic and secretory vesicles
