M1 L7: Translation and Genetic Code Flashcards
Bond between amino acids
Covalent, peptide bond
Biological pH amino acid zwitterion
molecule with +/- charges but no net charge
ammonium ~NH3+ and carboxylate ~COO-
What part of the ribosome makes the peptide bond
peptidyl transferase enzyme (ribozyme)
What are proteinogenic amino acids? How many are there? What’s the significance of non-proteinogenic amino acids? Example of one?
Amino acids found in biological proteins
20
Non-proteinogenic AAs have R groups similar to proteinogenic AAs –> cell may incorporate them into a polypeptide but they don’t function the same –> toxic
Canavanine
What are the ends of polypeptides? What ends of mRNA do they associate with?
N terminus –> 5’ end
C terminus –> 3’ end
Difference between a polypeptide and a protein?
Polypeptide = chain of many amino acids –> one polypeptide can be a protein
protein = functional structure (sometimes many polypeptides)
4 levels of protein organization and relevant bonds?
primary: sequence of amino acids, covalent peptide bonds
secondary: hydrogen bonding pattern of backbone (alpha helices or beta sheets), hydrogen bonds
tertiary: 3D shape after folding the polypeptide, ionic, covalent, hydrophobic
quaternary: 3D shape of many folded polypeptides (same forces)
What is protein structure ultimately defined by?
primary structure (sequence of AAs)
What are ribosomes made of?
rRNA and proteins
Ribosomes provide an environment for…
Peptide bond formation
Base pairing between mRNA and tRNA (codons and anticodons)
What direction to ribosomes read in? Where do they start and stop?
5’ to 3’ from start codon to stop codon
Purpose of the 5’ and 3’ UTRs?
Information for translation initiation and termination
Number of possible polypeptide sequences if n is the number of amino acids
20^n
3 key functions of ribosomes
bind mRNA and find start codon
facilitate mRNA/tRNA base pairing
catalyze peptide bond formation
What are the channels in the ribosomal subunits for?
Small subunit channel for mRNA
Large subunit channel for nascent polypeptide chain
How are rRNAs characterized?
Svedberg units (S)
- roughly proportional to size but not additive (S of small + S of large ≠ S of whole ribosome)
Cast of characters for bacterial translation initiation
- mRNA
- Small subunit
- Large subunit
- initiator tRNA with f-met
- initiation factors (IF3, IF2, IF1)
- GTP
Describe the steps of bacterial translation initiation
IF3 binds small subunit, helps bind mRNA, prevents large subunit from binding, base pairing between rRNA and Shine Dalgarno seq (3-9 bases upstream authentic start) *preinitiation complex
IF2 (bound to GTP) binds and facilitates initiator tRNA with f-met binding
IFI binds and prevents large subunit from binding
IF2 hydrolyzes GTP to GDP, energy makes large subunit bind
IF3, 2, 1 disassociate; A site open for tRNA
Cast of characters for eukaryotic translation initiation
- eIF1
- eIF1A
- eIF3
- eIF4 (a complex)
- eIF5
Describe the steps of eukaryotic translation initiation
eIF1, 1A, and 3 bind the small subunit and facilitate the next steps *preinitiation complex
initiator with tRNA and met binds
eIF5 binds, helps small subunit bind 5’ cap of mRNA
eIF4 binds, scans 5’ to 3’ for Kozak sequence (contains authentic start)
eIF5 hydrolyzes GTP to GDP, large subunit binds, eIFs disassociate, A site open
What’s different about archaean translation initiation? What question does it pose?
Some archaea have leaderless mRNA w/o a 5’ UTR
- ancestral state?
- did bacteria and eukaryotes gain 5’ UTR or did archaea lose 5’ UTR?
Cast of characters for translation elongation
- mRNA
- EPA sites
- charged tRNA
- EF-Tu bound to GTP
- EF-G
Describe the steps of translation elongation
BIND
1) EF-Tu (bound to GTP) binds to ribosome and helps charged tRNA bind to A site
BOND
2) codon/anticodon pairing between mRNA/tRNA leads EF-Tu to hydrolyze the GTP to GDP, EF-Tu disassociates
peptidyl transferase forms the peptide bond to the nascent chain –> nascent chain on tRNA in A site
MOVE
3) tRNA from P site moves to E, A moves to P, A site open
EF-G moves ribosome 3 bases towards 3’ end
Translation termination cast of characters
- Stop codons
- Release factors
Bacteria: RFI, RFII, RFIII
Eukaryotes: eRFI, eRFIII
archaea: aRFI
Describe the steps of translation termination
No tRNA can decode stop codons
RFI, RFII, eRFI, aRFI (bound to GTP) decode stop codon and bind the A site (bacteria need 2 release factors to decode all 3 stop codons)
Hydrolyze GTP, tRNA in P site releases polypeptide
RFIII, eRFIII help ribosomal subunits disassociate from mRNA (“recycling”)
What bacterial release factors decode which stop codons
RFI UAG and UAA
RFII UAA, UGA
What are polyribosomes
complex of many ribosomes which can decode one mRNA at the same time
How does the frequency of translation errors compare to transcription? How is this possible?
Translation has more errors. This is better tolerated because these mutations aren’t heritable. They only affect that one polypeptide or protein.
What are polycistronic mRNAs? What organisms are they most common in?
mRNAs with many genes on one transcript (each have own start and stop codons, are separated by intercistronic spacers)
common in bacteria
What are some post translational modifications? Why do we need them?
Bacteria cleave fmet
eukaryotes cleave N terminal met
phosphorylation, methylation
Important for making the protein functional
Why do we need the genetic code?
mRNA and amino acids are very chemically different, no great way to turn one into the other
Is the genetic code always universal? If there are exceptions, do they pose strong doubt for the single origin of life?
No, some organisms have changed the genetic code. Mostly in mitochondria - they have their own genome
Other exceptions are in yeast, all changed CUG (no longer encode for leucine)
Do not pose much doubt for single origin of life theory - changes are very slight. Genetic code mostly universal
Describe the different interpretations and actions tRNAs can make
interpret base pairing –> bind to A site, hydrolyze GTP, peptide bond
No base pairing –> leave A site, no hydrolysis or prptide bond
How can organisms encode 64 codons with less than 64 different tRNAs?
1) wobble hypothesis: 3rd base in pairing is relaxed
2) inosine modification: changing 3rd position A in tRNA to I (can ind with A, U, C) –> same tRNA can decode multiple codons
What charges tRNA
animoacyl tRNA synthetase adds amino acid to 3’ end of tRNA (acceptor stem)
very specific (multiple points of contact for tRNA), one for each of the 20 amino acids, ATP dependent
What are isoaccepting tRNAs
different tRNAs w/ same AA
Why did people think codons were 3 bases?
2 bases = 4^2 (4 nucleotides) = 16 codon combos (not enough for 20 AAs)
4 bases = 4^4 = 128 codons (way too many)
3 bases = 4^3 = 64 codons (reasonable)
3 questions about genetic code and how they were answered
Is it overlapping?
NO - point mutations don’t affect 3 adjacent AAs, no requirements for what AAs must follow each other
Does it have a delimiter? (spacers btwn codons)
NO - insertions/deletions shift whole frame
Is it made of triplets?
YES - 1 insertion/deletion messes up rest of AAs, 3 insertions/deletions near each other realigns rest of codons –> rest of AAs normal
H
How were codons deciphered?
In vitro
Decode single nucleotide codons first
Then make all possible codons, do 20 tests with each, each test has 1 tRNA w/ a radiolabeled AA
Add a ribosome, charged tRNAs, codons –> observe which radiolabeled AAs were not present after filtering the contents (tRNA w/ AA still stuck to ribosome, won’t pass through filter)
