MS-1 FUND 1: 1:00-2:00
Tuesday, September 23, 2014
Dr. David Schneider Protein Synthesis (1 of 2)
Abbreviations: DNA  deoxyribose nucleic acid, RNA  ribose nucleic acids, AA  amino acids
Transcriber: Kevin Jiang
Editor: Ryan Densmore
Page 1 of 4
Introductory Comments : This is the first hour of the two-hour lecture on Protein Synthesis by Dr. Schneider. The time stamps are based on ECHO recording
Protein Synthesis
Lecture begins at 5:40
ARS Question 5:40
I. Central Dogma of Biology (Slides 3-5) 6:15 a. Central dogma = DNA  RNA  Protein b. This is not a perfect representation though c. In reality, there are many shortcuts in between the processes and alternate pathways with other macromolecules like viral and cellular proteins (SN: Dr. Schneider said we will only view this from the linear pathway though) d. Central focus of the lecture is translation: RNA  Protein
II. Major Molecular Problem: How to convert nucleotide code to amino acid sequence? (Slides 6-7) 7:20 a. 4-bases DNA/RNA code has to code and provide instruction to make a protein from a pool of 20 amino acids b. Difficult because there is no direct chemical affinity between nucleotides and amino acids so requires a complex process i. No direct template for proteins c. Transcription vs. Translation i. Transcribing  like copying and involves the same “language” ii. Translation  have to change the document by converting it between “languages” iii. Translation in cells involve converting RNA nucleotide “language” to protein amino acid
“language”
III. Francis Crick Adaptor Hypothesis (Slides 8-9) 8:33 a. Francis Crick predicted the process of transcription and translation without any data i. Extraordinary because it’s difficult to synthesize models and hypotheses with data let alone without and still be relatively correct ii. Predicted the existence of mRNA, aminoacyl-tRNA, tRNA synthetase, editing by synthetase, etc. iii. Published his proposal in 1958 b. Crick’s Adaptor Hypothesis i. Crick thought amino acids were associated with specific adaptors that may contain nucleotides in them ii. The adaptors also recognized specific sequences in a RNA transcript which provided for the order of the amino acids in the polypeptide chain iii. Adaptors adapt the language of RNA to the language of proteins (now known to be tRNAs)
IV. Metaphor of Translation (Slide 10) 10:45 a. Genes are codes that are “recipes for proteins” b. RNA polymerase transcribes the code into a useful template c. Translation is the cracking of the code in collaboration with tRNA and ribosomes to make the protein d. We should appreciate how frequently and quickly this occurs in our cells with minimal error
V. Genetic Codons (Slides 11-12) 12:30 a. Nucleotide genetic code uses 3-base codons  64 possible combinations i. 4 total bases in DNA/RNA system so a triplet sequence has 4^3 (64) possibilities b. 2 base code only has 16 possibilities so it’s not enough to code for the 20 different AA’s c. 4 base code has too many possibilities (256) so can lead to error and variability in protein synthesis d. 3 base code is a good balance e. Each triplet is termed a codon and the corresponding matching sequence is the anti-codon f. Each codon references a specific AA or is a stop codon i. 61 of the codons code for an AA, 3 codons are stop codons and do not code for an AA ii. 3 stop codons: UAA, UAG, & UGA
1. Each stop codon codes for termination of translation iii. Not all amino acids have the same number of codons associated with them
3. Spectrum in between as well for the number of codons for an AA iv. AUG is the start codon and codes for Methionine
1. Some have up to 6 codons (arginine, leucine, and serine)
2. Some only have 1 codon (methionine and tryptophan)

MS-1 FUND 1: 1:00-2:00
Tuesday, September 23, 2014
Dr. David Schneider Protein Synthesis (1 of 2)
Abbreviations: DNA  deoxyribose nucleic acid, RNA  ribose nucleic acids, AA  amino acids
VI. tRNA Structure (Slide 13)
Transcriber: Kevin Jiang
Editor: Ryan Densmore
Page 2 of 4 a. tRNA is a relatively short RNA specie b. In contrast to most RNAs, tRNA is relatively stable and long lived due to its secondary structure i. rRNA is also very stable c. tRNA forms a 3 stem loop secondary structure called a clover leaf structure i. 2 lateral loops  T loop and D loop ii. 1 inferior anticodon loop that has the anticodon sequence that base pairs with the mRNA codon iii. 3’ acceptor end at the top that it bound to the AA residue
1. The 3’ acceptor end has a conserved CAA sequence
2. The terminal A covalently binds the AA d. The structure is called a clover leaf but it actually folds into a L-shape 3D structure in cells
VII. 3 Major Issues of Translation (Slide 15) 16:33 a. Translation process seems simple i. tRNA with AA attached will bind to the correct codon ii. tRNAs will put a bunch of AAs in a row based on the mRNA transcript and bind them all together to make amino acids iii. Not that simple though b. 3 major issues with this process i. Proper amino acid must be attached to every tRNA with high fidelity
1. Fidelity of the acceptor stem must be very good ii. tRNA anticodon needs to bind properly with the mRNA codon with high fidelity
1. Difficult because the codon involves relatively few bases so can be prone to error iii. Triplet reading frame has to be correct
1. Ribosome has to know where to start translating so to get the right reading frame
VIII. Problem #1: Charging the tRNA (ie. aminoacylation) (Slides 16-23) 18:25 a. Charging of tRNA/aminoacylation = adding AA to the tRNA b. Process uses specific tRNA synthetases i. Synthetase protein covalently binds the correct AA to th e 3’ acceptor stem of the tRNA molecule ii. Both the tRNA and the tRNA synthetase molecules are relatively small but this process needs to have high fidelity iii. Crystal structure imaging is really insightful in showing how the process works (SN: Slide 17 and
20 show this) c. tRNA synthetase shape is perfect to test key regions of the tRNA molecule to ensure specificity i. 3’ Acceptor site is buried into the protein active site ii. Active site is specific for the shape of the corresponding AA residue iii. T Loop and the Anticodon loop also interacts with the synthetase protein for recognition of correct tRNA before aminoacylation occurs d. 2 pathways of aminoacylation (Slide 18) 20:19 i. Divided into Class I and Class II aminoacyl-tRNA synthetases ii. Free AA’s are charged first by binding to AMP prior to aminoacylation occurs iii. Class I synthetases  facilitates nucleophilic attack by the 2 ’ –OH of the ribose ring of the terminal adenine in the 3’ acceptor site to the carbonyl carbon of the free AA-AMP
1. AMP is kicked o ff resulting in a 2’-O aminoacyl-tRNA
2. 2’- O aminoacyl-tRNA undergoes transesterification to form 3’-O aminoacyl-tRNA iv. Class II synthetases  facilitates nucleophilic attack by the 3’ –OH of the ribose ring of the terminal adenine in the 3’ acceptor site to the carbonyl carbon of the free AA-AMP
1. AMP is kicked off resulting in 3’- O aminoacyl-tRNA v. Two classes but the product is the same  3’ linkage aminoacyl-tRNA e. Fidelity of aminoacylation process is key to translational fidelity i. Slide 21 (22:07) shows a good example of this:
1. The size of the circles correspond to the consequence of mutation in that region to fidelity of translation a. Mutation in a region with large circle = major errors in translation ii. Regions are “identity elements” of the tRNA and key regions are:
1. 3’ acceptor stem region
2. Anticodon loop region
3. D – Loop region a. Some synthetases will sample this region even though it’s on the other side iii. Synthetases will associate with many tRNAs but have evolved the ability to recognize their specific tRNA through chemical interactions

MS-1 FUND 1: 1:00-2:00
Tuesday, September 23, 2014
Dr. David Schneider Protein Synthesis (1 of 2)
Abbreviations: DNA  deoxyribose nucleic acid, RNA  ribose nucleic acids, AA  amino acids
Transcriber: Kevin Jiang
Editor: Ryan Densmore
Page 3 of 4 f. tRNA synthetase editing (Slide 22) 23:48 i. Synthetase also has it’s own editing process to prevent error in aminoacylation ii. Synthetases have two active sites: synthesis site and editing site
1. Synthesis site is where the AA is bound to 3’ acceptor stem
2. Before the tRNA is released into circulation, the acceptor stem must pass through the editing site iii. Editing sites is highly specific for the correct AA
1. If it finds an incorrect AA, the editing site will cleave off the AA with an energetic reaction and then release the unbound tRNA
2. If the AA is correct, the editing site will allow release of the aminoacyl-tRNA
IX. Problem #2: How do appropriate tRNAs bind to the correct triplet codon (Slides 24-28) 26:45 a. Anticodon on the anticodon loop of the tRNA binds to the codon of the mRNA based on base pair affinity i. mRNA is 5’  3’ so the anticodon is 3’  5’ b. Base pairing occurs through hydrogen bonding i. Adenine:Thymine bonds have 2 hydrogen bonds ii. Glycine:Cytosine bonds have 3 hydrogen bonds
1. This is why G:C rich regions are harder to separate c. The first two base pairs in the codon bind with regular affinity d. The third base pair is called the wobble position i. Less selective than standard Watson-Crick base pairing e. Specific base pairing rules exist for the third position of the codon (Slide 26) 28:25 i. Base pairing is less specific in the third position so there’s more flexibility ii. tRNA has unique nucleotides like inosine
1. Inosine in the third position can bind uracil, cytosine, and adenine iii. Uracil normally can only bind to adenine
1. In the wobble position, it equally bind with guanine iv. Many other examples of this v. This could mean that you could have fewer tRNA species because of the flexibility of the wobble position
1. However, this is not the case (600 tRNA genes encoded in the human genome)
2. Cell haven’t exploited wobble to condense number of tRNA genes vi. Wobble does enable binding of the same anticodon to multiple codons
1. Ex. 3’-GUG-5’ anticodon can bind ‘5-CAC-3’ or 5’-CAU-3’
2. This is a reason why some AAs have multiple corresponding codons
X. Problem #3: The triplet code has to be read in the correct frame (Slides 29-31) 30:27 a. Ribosome must choose the correct reading frame on a massive mRNA to get the right sequence b. Even if the ribosome is off by 1 nucleotide sequence, it would shift the triplet code significantly i. Major mechanism of mutations as deletions or insertions cause frameshifts, which create nonsense sequences downstream of the mutation c. Solution: initiation of translation can only begin with a specific mRNA sequence i. Initiation can only start with the codon for methionine: AUG
1. Conserved across all species (bacteria, humans, etc.) d. Also there a strict 3-nucleotide transition in the elongation step that ensures the right reading frame is read
XI. Prokaryotic ribosome structure (Slide 35-45) 35:00 a. First crystal structure of the bacterial ribosome was only achieved 14 years ago b. Ribosome is composed of a large and small subunit i. 3 RNAs and 52 proteins involved ii. 2.5 million Da for total mass (50-80 thousand Da is the mass of the average protein) c. Functional parts of the ribosome with cartoon drawing (Slide 36-37) 35:35 i. Large subunit  teardrop-like shape ii. Small subunit  hamburger-like shape iii. mRNA sandwiched between the subunits iv. 3 functional domains made from the 2 subunits
1. E-site  exit site
2. P-site  peptidyl site
3. A-site  acceptor or active site v. Aminoacyl-tRNA enters at the A-site, it transfers is AA or AA chain on the P-Site, and the empty tRNA leaves on the E-site d. Transmission electron microscopy first enabled better visualization of the ribosomes (Slide 38)

MS-1 FUND 1: 1:00-2:00
Tuesday, September 23, 2014
Dr. David Schneider Protein Synthesis (1 of 2)
Abbreviations: DNA  deoxyribose nucleic acid, RNA  ribose nucleic acids, AA  amino acids
Transcriber: Kevin Jiang
Editor: Ryan Densmore
Page 4 of 4 i. Enabled scientists to interpret structures and hand draw out structures because there was no computer imaging back then ii. Visualized certain features on the ribosomes like the central protuberance that are still called that today e. Next step in imaging can from computer produced images in 1999 (Slide 39-43) i. Provided 3D models of the ribosomes but with relatively low resolution compared to todays technology ii. Features like the central protuberances were present and verified in these models iii. Also enabled additional visualization of the active sites (E-, P-, and A- sites)
1. Could visualize how the translation process worked in ribosomes iv. Crystal imaging in 2000 was important because it showed that no protein side chains were near the P-site
1. Meant that the rRNA functioned as the enzyme (ribozyme) in the translation process v. Also showed that the bulk of the ribosome comes from the RNA f. High resolution crystal structures (Slide 44-46) i. Showed the specificity of the A-, P- and E- sites ii. A lot of contact between the ribosome and the t-RNA in the ribosome structure iii. People who figures out this crystal structure won the Nobel Prize in 2009 g. Marat Yusupov did the first crystal structure of the eukaryotic ribosome in 2011 i. Figured out how to crystalize yeast ribosome ii. Enabled better understanding of antibody binding sites on the eukaryotic ribosome
XII. Differences between eukaryotic and prokaryotic ribosomes (Slide 48) 43:00 a. Eukaryotic ribosomes are a lot bigger and more complex i. Yeast ribosome is 3.3 million Da and human ribosome is 4.3 million Da compared to the 2.3 million Da prokaryotic ribosome ii. Most of the increased mass comes from insertion elements iii. Larger size is for better regulatory control b. Eukaryotic ribosomes vary across species more as well
Lecture ends at 44:20
Student Question 1: Will the tRNA synthetase check every one of the tRNAs that it processes?
Answer: The editing process is part of the release process for the tRNA synthetase so the whole system has pretty high fidelity.
<END OF LECTURE 59:59>