Protein Synthesis Flashcards
History of genetic code
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
Theoretical Consideration Predict a Non-overlapping triplet
- at least 3 bases must code for each amino acids
- non-overlapping triplet
Synthetic oligonucleotides helped to establish the relationship between codons and amino acids
- 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
Important features of genetic code
- 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
Reading frame
- 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
Single addition/deletion
- disturb reading frame
- often puts in a stop codon too early
- there is a truncated protein product
Point mutation
- single base changes can lead to amino acid changes
- not always that bad, sometimes is like in CF
Silent Mutations
- single base changes
- the nucleotide changes but the amino acid does not
tRNA molecule
- 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
Wobble hypothesis
- 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
Amino-Acyl tRNA synthetases
- 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)
Protein synthesis
-the process of protein synthesis is largely conserved from bacteria to man and that the remaining differences represent important target targets for antibiotics
Prokaryotic ribosome
- 70S ribosome
- 50S large subunit and 30S small subunit
Eukaryotic ribsome
- 80S ribosome is larger and contains proportionately more protein
- 60S large subunit, 40S small subunit
Ribosome can bind three tRNA molecules
-the ribosome has three binding sites for tRNA at the interface of the small and the large subunit
E= Exit
P= Peptidyl
A= Aminoacyl
Initiation
-mRNA binds and is alinged with respect to the correct reading frame; initiator tRNA binds; ribosome assembles from small and large subunits
Recognition of Reading Frame in initiation
- 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
Initiator tRNA
-special tRNA for initiation, is allowed to go straight to the P site
Initiation Factors
ancillary protein factors that associate transiently with components of the translation machinery to help in the assembly and disassembly of complexes
GTP
- 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
Steps of initiation
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
Elongation
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
Elongation Factors
- 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)
Elongation steps
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
Transpeptidation step
-does not require additional energy (amino acid tRNA is high energy) and the reaction is catalyzed by the large ribosomal subunit (ribozyme)
Termination
-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
Termination steps
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
Proofreading
- 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)
The Rate of Protein synthesis
- 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
High energy cost of protein synthesis
- 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
Regulation of Protein synthesis
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)
Regulation of translation by phosphorylation
-phosphorylation of eIF-2 leads to inhibition of translation
Regulating synthesis of globin in response to heme availability
- 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
Sequence elements within structure of mRNA regulate the translation of individual proteins
- 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
Regulation of Ferritin/Transferrin Receptor in response to iron availability
- 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)
Regulation of protein synthesis in plant and animal cells by micro and small interfering RNA molecules
- important way to regulate protein synthesis
- RNAi is a powerful too for knocking down gene expression in plants and animals
Antibiotics
- 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
Interferon
- 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
