Lecture 21: Protein Metabolism Flashcards
Translation Basics
Where does translation occur in Prokaryotes vs. Eukaryotes?
Prokaryotes Cells
- Transcription & Translation occur in the cytoplasm
Eukaryotic Cells
- Transcription occurs in the Nucleus
- Translation occurs in the cytoplasm
Genetic Code
How many nucleotides at a time does mRNA read?
3 nucleotides at a time
Genetic Code
How many different 3 letter combination possibilities are there?
How many possible reading frames are there
64 possible 3 letter combinations
3 possible reading frames
Genetic Code
What is a codon?
Triplet nucleotides that code for amino acid
Genetic Code
In what direction are codons read?
5’ -> 3’
Genetic Code
How many start codons are there?
Name them
1 - AUG
Genetic Code
How many stop codons are there
Name them?
3
UAA
UAG
UGA
Genetic Code
Is the genetic code universal?
Yes
It is universal among all living things with minor variations
Genetic Code
Is there redundancy present in the genetic code?
What is the purpose of redundancy?
Yes – Multiple different codons can encode the same AA
- Except for Met and Trp
The purpose of this is to minimize deleterious effects of mutations
Genetic Code
What is a frameshift mutation?
Deletions or insertions of 1 or 2 nucleotides
Genetic Code
What is the affect of a frameshift mutation
Changes the reading frame -> could be detrimental to protein function
Genetic Code
What are the essential components of translation?
- Codon
- tRNA
- Ribosome
Ribosomes
What do ribosomes do?
- Ribosomes are organelles found in all cells that synthesize proteins
- They are responsible for translating the genetic code in messenger RNA into a string of amino acids
- Those amino acids then form long chains that fold into proteins
Ribosomes
How do Ribosomes interact with tRNA?
Ribosomes have specific binding pockets for tRNA
Ribosomes
What are the binding pockets for tRNA?
EPA
E - Exit site
P - Peptidyl tRNA binding site
A - Aminoacyl- tRNA binding site
Ribosomes
How well are ribosome sequence and structure conserved?
Ribosome sequence and structure is highly conserved
Ribosomes
What are Eukaryotic Ribosomes made of?
80S subunit & 60S subunit
Ribosomes
What are Prokaryotic Ribosomes made of?
70S subunit & 60S subunit
tRNA
Describe tRNA in terms of it’s function in the translation process
tRNA is the adaptor molecule that reads and therefore decodes mRNA
tRNA
What does tRNA’s structure resemble?
What are the 4 arms on a tRNA molecule going clockwise
Which arms provide the backbone structure for the tRNA molecule itself?
it’s structure resembles a clover leaf
- Amino Acid arm
- T phi C arm
- Antocodon arm
- D arm
The T phi C arm and D arm
tRNA
What are anticodons?
What do they do?
The reverse complement of the mRNA strand
They base pair with the codons on the mRNA strand
tRNA
What occurs on the amino acid arm?
Where specifically are amino acids link to?
Specific amino acids are linked to the 3’ end
tRNA
What did Francis Crick notice that lead to the Wobble hypothesis
They noticed that the first two nucleotides in the codon: anti-codon predicted which amino acid would be coded
The tRNA did not fit perfectly – it wobbled
tRNA
What does the wobble hypothesis suggest?
It suggests that anticodon pairing isn’t completely precise
- If it was too specific -> protein synthesis would be too slow, dissociation not fast enough
tRNA
What does the wobble hypothesis say about how many unique tRNA are needed for all 61 coding codons
Only 32 unique tRNA are needed
- Different tRNA are needed only if any of the two nucleotides in a codon are different
tRNA
What are the 4 wobble pairs?
I-C
I-U
I-A
G-U
tRNA
How is I formed?
I is inosine and is formed from the deamination of adenosine
tRNA
What is the relationship between wobble and specificity?
More wobble = less specific
Less wobble = more specific
Decoding mRNA
What does the first codon in a sequence do?
Establishes the reading frame
Decoding mRNA
What is the importance of the AUG/start codon
Establishes the reading frame
- Translation continues until it encounters a stop codon
Mutations
What is a wild type?
The original/normal functional protein
Mutations
What is a substitution mutation?
What are the 3 types of substitutions?
A mutation that potentially leads to AA changes
- Silent
- Missense
- Nonsense
Mutations
Describe a silent mutation
no AA change due to wobble, retains function
Mutations
Describe a missense mutation
Mutations that lead to mistakes in the AA sequence
Mutations
Describe a nonsense mutation
Mutation that leads to premature termination, and no protein
Mutations
What is a frameshift mutation?
An insertion or deletion that shifts the reading frame
- Reading frame established by AUG start codon
Steps of Translation
What are the 5 steps of translation?
1) AA Activation
2) Initiation
3) Elongation
4) Termination/ribosome recycling
5) Folding/post translational processing
Steps of Translation
What is the main point of AA Activation?
tRNA are covalently linked to amino acids to for aminoacyl-tRNA
Steps of Translation
What does Aminoacyl-tRNA synthetase do?
Catalyze the formation of specific amino acids to specific tRNA
Steps of Translation
Describe Fidelity in the context of Translation
Nucleotides that are unique to a tRNA make specific contacts with a synthetase
Steps of Translation
What is the mechanism of AA Activation?
1) Amino acid is activated via adenylation
AA + ATP ⇌ Aminoacyl-AMP + PP
2) Aminoacyl-tRNA synthetase attaches AA covalently to tRNA
Steps of Translation
What is essential in Aminoacylation?
Accuracy is essential since amino acid identity is not checked again
Steps of Translation
Describe Class II synthetase that is utilized in AA Activation
3’ –OH directly attacks amino acid
Steps of Translation
Describe Class I synthetase that is utilized in AA Activation
2’ –OH attacks amino acid, which is then transferred to 3’ OH
Steps of Translation
How does the ribosome start scanning in a Prokaryotic vs. Eukaryotic cell?
Prokaryotic
- Shine-Dalgarno sequence on mRNA pairs w/ 16S rRNA and positions mRNA on ribosome
- Polycistronic: translate multiple genes at once
- Co-transcriptional (transcription and translation occur simultaneously)
Eukaryotic
- Ribosome scans staring from 5’ cap to find start (AUG) codon
- Monocistronic: translate one gene at a time
- NOT co-transcriptional
Steps of Translation
What are the steps of initiation in prokaryotes?
1) Small subunit (30S) and Initiation Factors (IF) assembles at the 5’ end of mRNA
2) Shine-Dalgarno sequence pairs with 16S portion of the small subunit
3) fMet-tRNA (initiator) binds to AUG in P site
4) IFs dissociate and large subunit (50s) completes ribosome complex. (full ribosome complex with initiator tRNA in P site)
Steps of Translation
What codes for fMet
Only initiator tRNA code for fMET, internal AUG utilize a different tRNA activated with MET
Steps of Translation
What is different about the mRNA of Eukaryotes vs. Prokaryotes?
Eukaryotic mRNA is capped and tailed
Steps of Translation
How is Initiation in Eukaryotes and Prokaryotes similar?
Small subunit and IFs bind at the 5’ end of mRNA
Steps of Translation
How is Initiation in Eukaryotes and Prokaryotes different?
- Initiator of tRNA binds and scans 5’ -> 3’ to find start codon (AUG)
- Starts with Met, no fMet
- No Shine-Dalgarno sequence
Steps of Translation
What are the steps in the Elongation phase of translation?
1) tRNA binds in A site with EF-Tu
(Elongation Factor)’
- GTP hydrolysis causes conformational causing EF-Tu to dissociate
2) Peptide bond formation
- Peptide chain now attached to tRNA in A site and grows out of the ribosome exit tunnel
3) Translocation
- tRNA without AA dissociates from E site
- tRNAs translocated/moved over via EF-G
Steps of Translation
What steps occur in termination?
1) A release factor (RF) is recruited and binds to A site with a stop codon
2) Peptide/AA-tRNA link is hydrolyzed, AA chain leaves
3) Components dissociate via GTP hydrolysis
Steps of Translation
What occurs in Folding and Post Translational processing
1) Unfolded is not yet functional, need to process and fold
2) Lots of processing/post-translational modifications possible
3) Folding happens spontaneously and co-translationally
Toxins
What do toxins and antibiotics do?
They inhibit translational machinery
Toxins
What are 3 toxins that inhibit protein synthhesis?
1) Puromycin
2) Tetracycline
3) Ricin
Toxins
How does Puromycin work?
Binds to A site, forms peptide bond, causes dissociation instead of translocation
Toxins
How does tetracycline work?
Also binds to A site but blocks tRNA binding
Toxins
How does Ricin work?
- Toxic protein against eukaryotic large subunit
- Depurates ribosome, destroys structure
Signal sequences and localization
How is cellular localization for proteins determined?
Proteins contain signal sequences to determine cellular localization
Signal sequences and localization
What are NLSs?
What do they do?
What type of cell might utilize them?
Some proteins contain nuclear localization signals (NLS), which directs their localization to the nucleus
Signal sequences and localization
What do alpha and beta importins do?
They bind to NLS and direct translocation through nuclear pores
Signal sequences and localization
What happens to Ran-GTP?
It is recycled due to GTP hydrolysis upon export
Protein Degradation
Why are proteins degraded?
They are degraded for lots of reasons
- only needed during specific times
- damage
Protein Degradation
What do bacteria, mitochondria, and chloroplasts utilize?
The Clp system, which uses ATP
Protein Degradation
What is ubiquitin?
Ubiquitin is a small protein that is covalently linked to proteins to tag them for degradation
The proteasome degrades ubiquinated proteins
