Translation

Question 1

mRNA

Answer 1

messenger RNA which contains coding sequence for the protein (template for protein synthesis)

Question 2

tRNA

Answer 2

transfer RNA, attaches to a specific amino acid at the ACCEPTOR STEM and to the mRNA at the ANTICODON LOOP

Question 3

How does the tRNA anticodon bind the mRNA codon?

Answer 3

5’ nucleotide of the anticodon on tRNA base pairs with the 3’ nucleotide of the codon in mRNA

Question 4

Ribosome

Answer 4

catalyzes protein synthesis, two subunits (large 60S and small 40S) made up of proteins and 4 ribosomal RNA molecules (rRNA)

Question 5

Translational Factors

Answer 5

other proteins that help in translational activation, initiation, elongation and termination

Question 6

Translation: Activation

Answer 6

THIS IS THE WHERE THE SPECIFICITY HAPPENS! 1. aminoacyl tRNA synthetases attach each amino acid to corrent tRNA at the amino acid’s C-terminus (1 phosphate bond)

Question 7

Translation: Initiation (3 steps)

Answer 7

THIS IS THE KEY REGULATORY STEP 1. initiation factors promote the association of Met-tRNA with 40S ribosomal subunit 2. other factors recognize mRNA cap and recruit 40S subunit to 5’ end of mRNA 3. 40S ribosomal subunit scans for AUG codon to start (2 phosphate bonds)

Question 8

Translation: Elongation (4 steps)

Answer 8

ribsome has 3 active sites: P-site (peptidyl), A-site (amino acyl-tRNA), E-site (exit) 1. tRNA+protein moves to P site 2. new amino acid moves into A site 3. peptide bond formed onto new tRNA/aa complex in A site 4. tRNA/aa complex moves into P site, next codon moves into A site (2 phosphate bonds overall)

Question 9

In what direction does ribosome read mRNA?

Answer 9

5’–>3’

Question 10

In what direction is protein assebled?

Answer 10

N terminus to C terminus (new bonds added to C terminus in growing chain)

Question 11

Polyribsome

Answer 11

one mRNA being simultaneously translated by many ribosomes

Question 12

Translation: Termination

Answer 12

release factors mimic tRNA shape and insert into A site when a STOP codon is present and induce peptidyl transferase to add H2O to the end of the chain

Question 13

Hallmarks of Prokaryotic Translation

Answer 13

1. transcription/translation are coupled 2. maller ribosomes (50S/30S) 3. initiates with f-Met amino acid 4. polycistronic mRNA

Question 14

Features of the Genetic Code

Answer 14

1. triplet code 2. degenerate (multiple codes for many amino acids (isoacceptors are different tRNAs for the same amino acid) 3. non-overlapping

Question 15

Start Codon

Answer 15

AUG

Question 16

Stop Codons

Answer 16

UAA, UAG, UGA

Question 17

missense mutation

Answer 17

one nucleotide change causes change in one amino acid

Question 18

silent mutation

Answer 18

one nucleotie change does not change the coded amino acid

Question 19

nonsense mutation

Answer 19

one nucleotide change creates a STOP codon

Question 20

frameshift mutation

Answer 20

an insertaion or deletion changes the reading frame from that point on

Question 21

Mitochondria translation

Answer 21

happens in the mitochondria (both transcription and translation), mostly producing rRNA, tRNA, and respiratory chain proteins

Question 22

Which part of the protein is used to target it for transport to the membrane?

Answer 22

the N-terminal signal sequence is recognized by Signal Recognition Particle (SRP)

Question 23

What does Signal Recognition Particle do?

Answer 23

SRP binds the N-terminus of an mRNA and stop elongation temporarily, SRP then binds SRP-receptor in endoplasmic reticulum (ER) which forms a pore for the nascent protein to enter once SRP releases and translation resumes

Question 24

What happens to proteins with no hydrophobic domains?

Answer 24

exuded into lumen of ER and will stay in ER, Golgi, lysosomes, or will be secreted from the cell (ex. Insulin, growth hormone, immunoglobins)

Question 25

What does mannose-6-phosphate do?

Answer 25

it is a post-translational modification added in the golgi to target proteins to the lysosome

Question 26

What happens to proteins that have other hydrophobic domains?

Answer 26

they will escape the transmembrane pore in the ER and move to a different spot in the membrane and will be moved to their destinatio by vesicle traffic

Question 27

How do vesicles fuse with their target membrane?

Answer 27

SNARE proteins on the vesicle and the traget membrane pair up and dock the vesicle close enough to the membrane for it to fuse (mediated by G-proteins (Rab-GTP) for directionality)

Question 28

Global regulation of translation

Answer 28

systematic increase or decrease of all mRNA translation at initiation step by phosphorylation

Question 29

eIF-2

Answer 29

global translation regulator, phosphorylation of eIF-2 decreases protein synthesis in response to exit from cell cycle, cell starvation, heat shock, or viral infection

Question 30

eIF-2 mechanism

Answer 30

1. active when bound to GTP, will bind 40S ribosomal subunit until it finds AUG, then GTP–>GDP and eIF-2 dissociates 2. if eIF-2 is phosphorylated it binds eIF-2B and becomes inactive (cannot start initiation => translation stops)

Question 31

eIF-4E

Answer 31

phosphorylation of eIF-4E stimulates protein synthesis in response to progression through cell cycle, growth factors and nutrients.

Question 32

eIF-4E mechanism

Answer 32

eIF-4E binds the 5’ cap on mRNA and helps recruit 40S ribosomal subunit to mRNA

Question 33

2 mechanisms for regulating eIF-4E

Answer 33

1. when not in use, eIF-4E is bound to 4E-binding protein and sequestered, if phosphorylated, eIF-4E will dissociate from 4E-BP (mTOR is the protein kinase that phosphorylates eIF-4E) 2. Salt bridge clamp, if stimulated by growth factors, it will be phosphorylated at different site by MNK1 that increases its affinity for mRNA and allows for repeated rounds of ribosome recruitment

Question 34

Sequence-Specific Regulation

Answer 34

regulation of individial mRNAs by sequence recognition, occurs at the level of mRNA stability or ribosome access

Question 35

Iron Homeostasis as an example of sequence-specific regulation

Answer 35

regulated by 4 factors 1. tranferrin carries iron in serum 2. transferrin receptor allows iron into cells 3. ferritin carries iron inside cells 4. aconitase/IRP-1 binds to iron response elements of mRNA strands to regulate translation/stability

Question 36

What happens to ferritin and transferrin receptors when iron is deficient?

Answer 36

aconitase is bound to iron response elements (IRE) on both mRNA strands and it promotes ferritin to not be made to prevent iron sequestration in cells, and transferrin receptors to be made to promote uptake of extracellular iron

Question 37

What happens to ferritin and transferrin receptors when iron is in excess?

Answer 37

aconitase released from both mRNA strands and causes ferritin to be translated to sequester iron inside cells, and transferrin receptor to not be made to keep iron out

Question 38

MicroRNAs as an example of sequence-specfic regulation

Answer 38

small RNAs (22-23 nucleotides) either suppress translation or destabilize specific mRNA with complimentary sequences

Question 39

How are miRNAs made?

Answer 39

RNA pol 2 transcribes primary miRNA which are processed (5’ cap, splicing, 3’ tail) and then form stem-loop structures bc of intramolecular H-bonds. The Pri-miRNA is trimmed by Drosha and transported to the cytosol where it is trimmed into 22-23 nt peices

Question 40

How important isit for miRNA to be exactly complementary?

Answer 40

imperfect sequence match = suppress translation, prefect sequence match = cleavage and degradation by RISC complex (called RNA interference)

Question 41

What are the current hurdles in miRNA therapy development?

Answer 41

1. delivering miRNA to the appropriate target cell 2. cost!