Protein Synthesis

Question 1

History of genetic code

Answer 1

1950- A content equal to T, C to G
1952- DNA carries genetic information
1953- Structure of DNA
1955- Adapter hypothesis
1961- Triplet nature of codons
1961-First codon determined
1965- complete code availible
1964- nucleotide sequence of tRNA
1971- central dogma of molecular biology
1972- basic understanding of translation

Question 2

Theoretical Consideration Predict a Non-overlapping triplet

Answer 2

• at least 3 bases must code for each amino acids

- non-overlapping triplet

Question 3

Synthetic oligonucleotides helped to establish the relationship between codons and amino acids

Answer 3

• the genetic code was deciphered
• the polarity of translation was determined (goes from 5’ to 3’
• within a year the entire genetic code was determined

Question 4

Important features of genetic code

Answer 4

• knowing the genetic code, one can theoretically deduce the protein sequence that will be synthesized from any given DNA sequence
• the genetic code is almost universal (exception: mitochondrial DNA)
• the code is degenerate (contains synonyms)
• the code is not random
• Start codon- AUG (met)
• Stop codon- UAA, UAG, UGA

Question 5

Reading frame

Answer 5

• 1st reading frame UAC UAC UAC UAC
• 2nd reading U ACU ACU ACU ACU
• 3rd reading frame UA CUA CUA CUA C

-code is read in triplets following the initiator AUG

Question 6

Single addition/deletion

Answer 6

• disturb reading frame
• often puts in a stop codon too early
• there is a truncated protein product

Question 7

Point mutation

Answer 7

• single base changes can lead to amino acid changes

- not always that bad, sometimes is like in CF

Question 8

Silent Mutations

Answer 8

• single base changes

- the nucleotide changes but the amino acid does not

Question 9

tRNA molecule

Answer 9

• a cloverleaf secondary structure, stabilized by Watson-Crick base pairing
• base-pairing between the 5’ and 3’ ends forms the acceptor or amino acid stem, the stem has the nucleotides -CCA-OH at its 3’ end, which is where the amino acid will be attached by a specific amino-acyk tRNA synthetase
• an anticodon loop in the middle that interacts with the codon of mRNA
• a complex teritiary structure maintained by hydrogen bonding and stacking of bases that results in an overall L-shape with the anticodon on one end and acceptor stem at the other end

Question 10

Wobble hypothesis

Answer 10

• there is not a separate tRNA for every codon
• suggests that the first two bases of the codon: anticodon interaction are constrained by normal Watson-Crick base-pairing, but that the requirements for hydrogen bonding at the third bases is less stringent
• means some tRNAs can recognize more than one codon

Question 11

Amino-Acyl tRNA synthetases

Answer 11

• enzymes that link to amino acids to their corresponding tRNAs
• transfer of a specific amino acid to the 3’ OH of specific tRNAs
• the amino acid is attached to the tRNA via its C=terminus and at the 3’ end of the tRNA

ATP + amino acid +tRNA –> aminoacyl-tRNA +AMP + PPi (PPi -> 2 Pi)

Question 12

Protein synthesis

Answer 12

-the process of protein synthesis is largely conserved from bacteria to man and that the remaining differences represent important target targets for antibiotics

Question 13

Prokaryotic ribosome

Answer 13

• 70S ribosome

- 50S large subunit and 30S small subunit

Question 14

Eukaryotic ribsome

Answer 14

• 80S ribosome is larger and contains proportionately more protein
• 60S large subunit, 40S small subunit

Question 15

Ribosome can bind three tRNA molecules

Answer 15

-the ribosome has three binding sites for tRNA at the interface of the small and the large subunit
E= Exit
P= Peptidyl
A= Aminoacyl

Question 16

Initiation

Answer 16

-mRNA binds and is alinged with respect to the correct reading frame; initiator tRNA binds; ribosome assembles from small and large subunits

Question 17

Recognition of Reading Frame in initiation

Answer 17

• start translation at AUG
• Shine-Dalgarno Sequence in prokaryotes, the ribosome binds interior to the message- bacteria have polycistronic messages

-eukaryotes- mRNA has a 5’ cap at the 5’ untranslated region (5’-UTR)- monocistronic

Question 18

Initiator tRNA

Answer 18

-special tRNA for initiation, is allowed to go straight to the P site

Question 19

Initiation Factors

Answer 19

ancillary protein factors that associate transiently with components of the translation machinery to help in the assembly and disassembly of complexes

Question 20

GTP

Answer 20

• cycles of translation factor association and dissociation, often coupled with GTP hydrolysis, ensure that conformational changes in the ribosome occur in the forward direction
• GTP binding and hydrolysis can convert proteins between active and inactive conformations

Question 21

Steps of initiation

Answer 21

1) 30S initiation complex- eIF-2-GTP-initiator tRNA (like tRNAiMet) and mRNA bind to small subunit
2) GTP hydrolyzed, releasing eIF-2-GDP and driving the assembly of the large ribosomal subunit. Initator tRNA at P site

Question 22

Elongation

Answer 22

Amioacyl tRNA binds and checks codon-anticodon match, new peptide is formed, growing chain is translocated from A-site to P-site, and mRNA is pulled along so that next codon is exposed to A-site

Question 23

Elongation Factors

Answer 23

• one to bring each aa-tRNA in and check the codon-anticodon match (EF-Tu)
• one to help shift the mRNA and tRNA by three nucleotides to prepare for the next aa-tRNA (EF-G)

Question 24

Elongation steps

Answer 24

1) EF-Tu forms a complex in the cytosol with GTP and aminoacyl-tRNA
2) Complex binds to the ribosome, kicking out the tRNA in the E site, GTP is hydrolyzed to GDP and the aminoacyl-tRNA is left bound to A site of ribosome as EF-Tu, now complexed with GDP dissociates from ribosome
3) formation of peptide bond, transpeptidation- peptidyl transferase
4) translocation- uncharged tRNA left in the Psite moves to the E site. tRNA with attached peptide (peptidyl tRNA) is translocated from the A- site to the P-site and the mRNA is pulled along with tRNA
5) Growing polypeptide chain attached to tRNA in P-site, mRNA moved by 3 nucleotides so that anew codon is exposed, A site is empty, E site contains spent tRNA. Cycle continues until STOP codon is reached

Question 25

Transpeptidation step

Answer 25

-does not require additional energy (amino acid tRNA is high energy) and the reaction is catalyzed by the large ribosomal subunit (ribozyme)

Question 26

Termination

Answer 26

-release factors bound to GTP bind to stop codon in A-site, peptidyl tRNA in P-site is hydrolyzed to release peptide chain and leave tRNA in P-site. tRNA, release factors, and mRNA are released from ribosome after GTP hydrolysis

Question 27

Termination steps

Answer 27

1) release factor binds A-site 2) peptide is hydrolyzed and released- water molecules take role of incoming tRNA (ester bond between peptide and 3' end of P-site tRNA is hydrolyzed) 3) Components dissociate after GTP is hydrolyzed to GDP and Pi changing conformation of ribosome

Question 28

Proofreading

Answer 28

- aminoacyl tRNA synthetase has two active sites for synthesis and editing - each individual site prodvides moderate specificity - the overall specificity is the product of the individual specificities - incorrect amino acid costs two phosphoanyhdride bonds - incorrectly base-paired tRNAs dissociate before transpeptidation (which is irreversible)

Question 29

The Rate of Protein synthesis

Answer 29

- the rate of protein synthesis is 15-20 amino acids per second in bacteria and 7-9 amino acids per second in eukaryotes - one of the rate limiting steps in protein synthesis bty the ribosome is hydrolysis of GTP bound to Ef-Tu - proofreading during elongation slows down overall rate but is essential for high fidelity of translation

Question 30

High energy cost of protein synthesis

Answer 30

- charging tRNA- two phosphoanhydride bonds (ATP to AMP + 2Pi) - inititation- one GTP to bring in initiator tRNA unknown number of ATPs to scan for AUG - elongation- two GTP/amino acid incorportated - termination- one GTP when polypeptide is released - plus- unknown amount of energy needed for proofreading and proper folding

Question 31

Regulation of Protein synthesis

Answer 31

1) Regulation of translation at the level of initiation via controlling phosphorlyation of eIF-2 Example: Regulating the synthesis of globin in response to heme availability 2) Sequence elements within the structure of mRNA regulate the translation of individual proteins Example: Regulation of Ferritin/Transferrin Receptor 3) Regulation of protein synthesis in plant and animal cells by micro and small interfering RNA molecules (miRNA and siRNA) Example; RNA interference (RNAi)

Question 32

Regulation of translation by phosphorylation

Answer 32

-phosphorylation of eIF-2 leads to inhibition of translation

Question 33

Regulating synthesis of globin in response to heme availability

Answer 33

- occurs by inhibition of translation when heme is not availible - in the absence of heme, cells activate a protein called HCI (heme-controlled inhibitor) which phosphorylates eIF-2 - when heme becomes availible again, HCI is inactivated, the phosphate removed, and eIF-2 can be recycled

Question 34

Sequence elements within structure of mRNA regulate the translation of individual proteins

Answer 34

- the lifetime of mRNA is important in the regulation of protein synthesis - if poly A tail is too short- 5' cap is then removed and there is both 5' to 3' and 3' to 5' degredation

Question 35

Regulation of Ferritin/Transferrin Receptor in response to iron availability

Answer 35

- translation of the mRNA encoding ferritin, an intracellular iron storage protein, is increased rapidly if the concentration of iron within the cell increases - at the same time translation of the mRNA encoding the transferrin receptor, which imports iron, is decreased - effects are mediated by aconitase which in the absence of iron, binds to specific stem-loop structure (IRE)

Question 36

Regulation of protein synthesis in plant and animal cells by micro and small interfering RNA molecules

Answer 36

- important way to regulate protein synthesis | - RNAi is a powerful too for knocking down gene expression in plants and animals

Question 37

Antibiotics

Answer 37

- antibiotics interfere with prokaryotic protein synthesis - they have diverse chemical structures due to the diversity of their targets - small molecules that inhibit translation have provided important information to help elucidate the mechanism of protein synthesis

Question 38

Interferon

Answer 38

- secreted by cells in response to viral infection involving dsRNA - intercellular messengers that tell other cells to shut down protein synthesis to prevent spreading of a viral infection