Exam 2 10/13 Britton Flashcards
List 2 gene regulatory mechanisms in which RNA stability is affected
- deadenylation-dependent mRNA decay
- microRNA (miRNA)
What does deadenylation nuclease (DAN) do?
- Degrades mRNA
- associates with mRNA 5’ methyl cap and degrades the mRNA in the 3’ to 5’ direction
Deadenyltion nuclease degrades mRNA in the __ to __ direction
3’ to 5’
In addition to DAN, the _____ also uses the mRNA 5’ cap and 3’ polyA tail, so there is _____
translation initiation apparatus; competition
What are miRNA?
small single stranded RNA molecules that negatively regulate gene expression
miRNA have ____ base pairing to how many target mRNA transcripts?
perfect/near perfect; one or several (rarely just 1 target)
What happens when miRNA bind to specific sequences in target mRNA?
- promotes cleavage of the target mRNA
- base pairs with target mRNA to negatively regulate its expression (inhibits protein translation of the target mRNA)
_____ degrades mRNA
Deadenylation nuclease
True or false: some miRNA is translated into protein
False - they are not translated
What do miRNA gene sequences include?
- mRNA target sequence
- approximate reverse complement sequence
Steps of miRNA processing following transcription
- primary mRNA forms hairpin loop structure
- pri-miRNA is processed into pre-miRNA (shorter loop structure)
- further processing and cleavage (Drosha and Dicer enzymes) to produce mature miRNA
- miRNA loaded onto RNA Induced Silencing Complex (RISC), includes Argonaute proteins
- Directed towards target mRNA
____ plays a role in osteogenesis
miRNA
miRNA plays a role in diseases such as:
- Heart development and disease
- diabetes
- cancer
How does miRNA play a role in heart development?
Regulation of specific transcription factors important for cardiogenesis
How does miRNA play a role in diabetes?
- lack of miR-375 causes b cells (insulin producing) to die
- a cells (glucagon producing) increase in mass
miRNA can act as ____ to aid cancer treatment
Biomarkers
Translation of mRNA is carried out on:
Ribosomes
____ serve as adaptors between the mRNA template and the amino acids incorporated into protein
tRNAs
Protein synthesis involved interactions between ____ types of RNA molecules
3 (rRNA, tRNA, mRNA)
Ribosomes are known as the:
translational apparatus
Where are ribosomes located?
- free floating in the cytosol
- associated with the ER
Ribosomes consist of ____ subunits
2 (large and small)
mRNA is associated with which ribosomal subunit?
Small
What does each ribosomal subunit consist of?
- rRNA
- several ribosomal proteins
Ribosome has ___ binding sites
3 tRNA binding sites - A, P, E
What happens at ribosome A site?
aminoacyl-tRNA enter at A site
What happens in the P site of ribosome?
peptidyl tRNA is bound
(tRNA with growing peptide chain attached)
What happens at E site of ribosome?
deacylated tRNA exit at E site
What direction is mRNA translated?
5’ to 3’
Codon
Groups of 3 nucleotides on mRNA
Initiation codon
AUG
____ sets the reading frame for protein translation
Initiation codon AUG
How many possible codons are there, and how many code for amino acids?
64 total possible; 61 code for amino acids
___ codons are stop codons
3
What is meant by degeneracy of the code?
Most amino acids are represented by more than one codon (ex. leucine, serine)
During translation, tRNA links the codon on mRNA transcript to a _______
specific amino acid
2 key sites on tRNA
- anticodon
- amino acid attachment site at 3’ end
Complementary base pairing with mRNA occurs at:
Anticodon (located on tRNA)
The 3’ end of tRNA is the site of:
Attachment of specific amino acids
True or false: there are 64 tRNAs that have distinct anticodons
False - Wobble in the 3rd position allows for the anticodon of one tRNA that can bind with several different codons
The genetic code is translated in a ___ step process that uses energy from ____
2; ATP
Translation control steps
- specific amino acid linked to specific tRNA
- tRNA (anticodon) binds to mRNA codon by complementary base pairing
Each aminoacyl tRNA synthetase is selective for:
Particular amino acid
How is specificity of aa attachment to tRNA controlled?
Proofreading reactions will hydrolyze any incorrectly formed aa-tRNAs
Translation occurs in 3 stages which are:
- initiation
- elongation
- termination
What happens during initiation of translation?
- synthesis of the first peptide bond of protein
- ribosome needs to bind to mRNA to form initiation complex (1st AUG-tRNA)
What happens during elongation of translation?
chain is extended by the sequential addition of amino acids
What happens during termination of translation?
- aa addition is stopped
- completed protein is released and dissociates from the mRNA
_____ is optimal for translation initiation
Kozak sequence
What can block translation initiation?
- translation repressor proteins can hide the Kozak sequence
- miRNA
What is the Kozak sequence?
A/G-X-X-A-U-G-G
First AUG codon from 5’ cap site on mRNA
Start codon
What is the function of eukaryotic initiatior factors (eIF)?
Control mRNA translation
eIF-2/4
If mRNA is _____, eIF-4 will associate with mRNA transcript at _____
correctly processed; 5’ cap and 3’ polyA tail
Once eIF-4 associated with mRNA, what happens?
mRNA can be correctly positioned in the small ribosomal subunit
eIF-2 binds to:
initiator tRNA molecule
Translation initiation: The ____ is brought to the small ribosomal subunit in association with ____
initiator-tRNA; eIF-2
True or false: The initiation tRNA can only bind when the small ribosomal subunit is not attached
False - if large is not attached
The initiator-tRNA scans the mRNA transcript looking for:
Kozak sequence (AUG start codon)
eIF2/4 must ____ to allow the large ribosomal subunit to bind and begin translation
dissociate
What causes eIF proteins to dissociate?
GTP-eIF2 is hydrolyzed to form GDP, causing dissociation
How is eIF-2 activity controlled?
- association with guanine nucleotide exchange factor eIF-2B
- should be phosphorylated
Phosphorylated eIF-2 is active or inactive? Would protein synthesis be faster or slower?
Inactive and sequesters eIF-2B as an inactive complex
Protein synthesis is slowed
If eIF-2 is phosphorylated ____ cannot participate in GDP to GTP exchange
eIF-2B
Steps of translation initiation associated with eIF-2
- initiatior-tRNA brought to small ribosomal subunit in association with eIF-2 (GTP bound)
- initiatior-tRNA scans mRNA transcript in search for Kozak sequence (AUG start codon)
- eIF-2 GTP is hydrolyzed to form GDP, allowing eIF-2 (and 4) to dissociate
- Large ribosomal subunit can bind and begin translation
Steps of translation elongation phase
regarding each binding site
- aa-tRNA binds to vacant A site (enters)
- new peptide bond formed at P site
- mRNA moves 3 nucleotide positions through the ribosomal subunit to the E site
- tRNA is used up and ejected
EF-1 and EF-2
- Elongation factors
- facilitate peptide chain elongation during protein synthesis
Function of EFs (elongation factors)
- control the accuracy of translation (and proofreading)
- drive the elongation phase of translation forward to make it faster
EF-1 transports aa-tRNAs into the ___. After peptide bond formation, EF-2 mediates ____
A site; ribosome translocation and resetting
EF-1 and EF-2 are associated with what source of energy?
GTP/hydrolysis to GDP
EF-1 and EF-2 use the hydrolysis of GTP to GDP to:
- displace any incorrectly base-paired tRNA
- reset the ribosome
- eject the spent tRNA
Post-translational control of gene expression refers to the control of:
Protein activity, stability, localization, and interacting partner molecules
Proteins can be modified to make them:
Functionally active/inactive
True or false: There are both reversible and irreversible events of protein modifications
True
Proteolysis is reversible or irreversible?
Irreversible
Post translational modifications are reversible or irreversible?
Reversible
A protein’s ____ depends on its shape
Function
During protein folding, what structures usually form first?
- alpha helix
- beta sheet
secondary protein structure
Example of protein with quaternary structure
Hemoglobin
Alpha helices and beta sheets within a protein can interact to form:
Tertiary structure
Proteins fold into ____ shapes which include forming _____ to bind a particular molecule
Functional; pockets
Lock and key
Failures in protein ___ causes several known diseases
folding
Cystic fibrosis and sickle cell anemia are associated with
Mutations that cause protein misfolding
Accumulation of mis-folded proteins play a large role in _____ diseases such as:
Neurological; Alzheimer’s, Parkinson’s, Lou Gehrig’s disease (ALS), Huntington
Disulfide bridge
Linking of sulfur residues between two cysteine aa’s
Preproinsulin
Precursor of insulin, has A and B chain joined by intervening C chain
How is preproinsulin modified to form insulin?
- C chain removed by cleavage
- A and B chains connected by disulfide bonds
Examples of post translational modifications
- methyl
- acetyl
- hydroxyl
- phosphate
- ubiquitin
- lipids
- carbohydrates
Where do post translational modifications occur?
- cytoplasm
- nucleus
- Golgi
Proteins can be activated or deactivated through the activity of
Kinases and phosphatases
Phosphorylation can happen on amino acids which have a ___ residue, which are
hydroxyl; serine, threonine, tyrosine
Ubiquitination
Regulated degradation mechanism to control protein levels; adding ubiquitin is a PTM
Ubiquitin is found in
Most tissues
Ubiquitination steps (3)
- Activation via ubiquitin-activating enzymes (E1)
- Conjugation by ubiquitin-conjugating enzymes (E2)
- Ligations by ubiquitin ligases (E3)
Amino Can’t Live
Ubiquitination marks proteins for _____ via _____
Degradation via proteosome
Degradation of cyclins, TFs, protein kinases occurs through:
Ubiquitination
Rough ER has ribosomes on
cytosolic surface
In post-translational transport, where does translation occur?
In ribosomes in cytosol
In post-translational transport, once a protein is made, it is transported to
Its functional cellular location (nucleus, mitochondria, peroxisomes, etc)
In co-translational transport, ribosomes with mRNA attached are targeted to the ___, where translation occurs in association with the
ER; ER
Ribosomes are targeted to the RER by a ____ at the ____-terminal of the translated protein
signal sequence; N-terminal
What is a signal sequence
short stretch of amino acids, interacts with SRP (signal recognition particle)
If a protein does not have a signal sequence, where does it go?
In the cytosol and translated there
Proteins with a signal sequence move from the RER to:
Golgi, then secretory vesicles, plasma membrane, lysosome
What is the signal hypothesis?
- signal sequence synthesized and binds to SRP
- SRP binds to receptor on RER
- Protein synthesized into ER
- Signal is removed when the growing peptide enters the ER
Protein synthesis is complete
Constitutive secretion
proteins secreted from a cell continuously regardless of external factors or signals
Regulated secretion
Proteins secreted from a cell when a specific signal is detected by the cell (insulin secretion)
The ____ is the major site for the modification and packaging of proteins
Golgi
Different coat proteins select different _____ and determine different _____
protein cargo; transport between ER, Golgi, organelles, cell membrane
Coat proteins examples
Clathrin
COP1
COPII
Once a coated vesicle finds a target membrane, they
Fuse; exocytosis
SNARE stands for
Soluble NSF Attachment protein Receptors
v-SNAREs
vesicle membrane SNAREs
t-SNAREs
target membrane snares
Complementary ______ proteins form stable complexes to allow for fusion
v-SNAREs and t-SNAREs
True or false: each complementary set of v-SNAREs and t-SNAREs is associated with a particular organelle involved in the secretory pathway
True - example neurotransmitter release
