Translation Flashcards
What explains the difference between the numbers of codons and the types of tRNAs found in eukaryotic cells?
There are far more codons than types of tRNAs; not every tRNA will have an anticodon that complements the codon found in a cell. Wobble at the 5’ nucleotide in an anticodon allows a single tRNA charged with a particular amino acid to recognize several or even all of the codons (at the 3’ end) for that amino acid.
What are the events of translation initiation, elongation and termination?
- tRNAs are charged by amino-acyl-tRNA synthetases, which covalently attach amino acids to the tRNA’s 3’ end to form amino-acyl-tRNAs.
Initiation - The small ribosomal subunit binds first to the methylated cap at the 5′ end of the mature mRNA.
- The small subunit then migrates to the initiation site—usually the first AUG it encounters as it scans the mRNA in the 5′-to-3′ direction.
Elongation - Elongation factors usher the charged tRNA into the A site.
- Peptidyl transferase forms a peptide bond between the initiating tRNAi at the P site and the new tRNA at the A site.
- The ribosome moves and exposes the next mRNA codon. Simultaneously, the initiating tRNA (which no longer carries an amino acid) moves to the E site and the tRNA carrying the dipeptide at the A site moves to the P site.
- The empty A site receives a new amino acid, specified by a codon. Simultaneously, the tRNA at the E site is kicked off.
- Peptidyl transferase forms a peptide bond between the tRNAs at the P and A site again.
Termination - The stop codon moves into the A site and is recognized by release factors.
- The completed polypeptide chain is separated from the tRNA via release factors.
- This tRNA, the mRNA, and the large and small subunits of the ribosome then dissociate from each other.
What are the steps in initiation?
- The small ribosomal subunit binds first to the methylated cap at the 5′ end of the mature mRNA.
- The small subunit then migrates to the initiation site—usually the first AUG it encounters as it scans the mRNA in the 5′-to-3′ direction.
What are the steps in elongation?
- Elongation factors usher the charged tRNA into the A site.
- Peptidyl transferase forms a peptide bond between the initiating tRNAi at the P site and the new tRNA at the A site.
- The ribosome moves and exposes the next mRNA codon. Simultaneously, the initiating tRNA (which no longer carries an amino acid) moves to the E site and the tRNA carrying the dipeptide at the A site moves to the P site.
- The empty A site receives a new amino acid, specified by a codon. Simultaneously, the tRNA at the E site is kicked off.
- Peptidyl transferase forms a peptide bond between the tRNAs at the P and A site again.
What are the steps in termination?
- The stop codon moves into the A site and is recognized by release factors.
- The completed polypeptide chain is separated from the tRNA via release factors.
- This tRNA, the mRNA, and the large and small subunits of the ribosome then dissociate from each other.
What direction does the polypeptide chain grow in?
N terminus to C terminus (new amino acids added at the C terminus)
What makes the initiating tRNAi different from the other tRNAs?
It enters the P site of the ribosome. The others all enter at the A site.
It carries unaltered methionine, the others all carry different amino acids.
What factors besides ribosomes are necessary for translation initiation?
- The 40s ribosomal subunit associates with eIF (eukaryotic initiation factors) and the Met-tRNA.
- mRNA associates with the eIF (and PABPC).
- mRNA and 40s subunit associate with one another.
- 40s subunit scans for and finds AUG codon.
- eIFs help 60s subunit to join the 40s subunit.
- Mature 80s subunit begins the translation.
What are two examples of ribosomopathies and what are their genetic bases?
- Diamond-Blackfan anemia
There are 4-5 cases/million live births and this requires a bone marrow transplantation for survival.
This is due to mutations in either the RPS or RPL genes that deplete the number of 40s/60s subunits, leading to fewer mature ribosomes. - Treacher Collins Syndrome
This is a mandibulofacial dysostosis, affecting 1/10,000-50,000 live births. It leads to problems with the airway, swallowing, brain development, and hearing and causes extensive craniofacial problems.
The majority (93%) have a mutation in TCOF1, which localizes to the nucleolus, which interacts with ribosomal DNA and recruits RNA pol I to the nucleolus.
The remaining 6% have mutations with POLR1C or POLR1D, which are RNA pol I genes that transcribe rRNA other than the 5S rRNA.
Describe Diamond-Blackfan anemia. What causes it?
There are 4-5 cases/million live births and this requires a bone marrow transplantation for survival.
This is due to mutations in either the RPS or RPL genes that deplete the number of 40s/60s subunits, leading to fewer mature ribosomes.
Describe Treacher Collins syndrome. What causes it?
This is a mandibulofacial dysostosis, affecting 1/10,000-50,000 live births. It leads to problems with the airway, swallowing, brain development, and hearing and causes extensive craniofacial problems.
The majority (93%) have a mutation in TCOF1, which localizes to the nucleolus, which interacts with ribosomal DNA and recruits RNA pol I to the nucleolus.
The remaining 6% have mutations with POLR1C or POLR1D, which are RNA pol I genes that transcribe rRNA other than the 5S rRNA.
What do RPS19, RPS24, and RPS17 genes do?
produce 18S rRNA, which is needed to produce 40s subunits
What do RPL5, RPL11, and RPL35A genes do?
produce the 60s subunit
What does the TCOF1 gene do?
encodes treacle protein, which localizes to the nucleolus, interacts with ribosomal DNA, and recruits RNA pol I to the nucleolus
What do POLR1C and POLR1D genes do?
RNA pol I genes that transcribe rRNA other than 5S rRNA
Many (but not most) genes have mRNAs with upstream open reading frames in their 5’ UTR. What is a commonly accepted function of these upstream ORFs?
uORFs reduce translational output for mRNAs that encode growth factors. This reduction in protein is normal and important for regulating cell differentiation (ex. in the nervous system).
What observations help us understand the function of upstream ORFs?
5’UTR of PTCH1 was hooked to a fluorescent reporter gene. When 3 uORFs were removed, 5 fold more fluorescent protein was made. When 1 uORF mutated, fluorescence intensity doubled.
This shows that uORFs in 5’ UTR regulate by making translation LESS efficient.
What kinds of genes seem to have upstream ORFs?
Developmental genes; genes that are very potent, which can easily make too much protein
How do uORFs work?
The 40s subunit binds to the methylated 5’ cap, then searches for the initiator codon. If it finds an uORF first, the ribosome will dissociate and no effective protein will be made.
How do microRNAs contribute to regulating transcript and protein abundance?
21-24 nt noncoding miRNA guides bind to mRNAs, key regulators of mRNA abundance
How does complementarity between mRNA and miRNA determine the fate of the mRNA?
If there is perfect complementarity, the mRNA will be targeted for degradation.
If there is imperfect complementarity, the mRNA is not translated. The miRNA causes the ribosomes to leave, then interacts with eIFs in charge of ribosomal creation.
Explain how cells remove transcripts that have premature termination codons or other kinds of damage that might result in defective proteins.
Within the nucleus, the TRAMP complex recognizes defective transcribed RNA and targets it for degradation inside of the nucleus.
Within the cytoplasm, if the mRNA lacks a stop codon, non-stop decay occurs. The ribosome keeps translating, leading to the addition of multiple wrong amino acids.
mRNA abundance is relatively easy to measure and this can even be done at large scale by single cell RNA-sequencing, in which transcripts for thousands of genes are isolated and counted for every cell separately from a tissue or embryo. By contrast, protein quantification is much more challenging, and especially so at single-cell level. What are the risks of inferring gene function from mRNA abundance data alone?
There is no correlation between the amount of mRNA and the amount of protein that is produced.
mRNA abundance is relatively easy to measure and this can even be done at large scale by single cell RNA-sequencing, in which transcripts for thousands of genes are isolated and counted for every cell separately from a tissue or embryo. By contrast, protein quantification is much more challenging, and especially so at single-cell level. How predictive are such data for estimating protein abundance and why?
What is eIF?
eukaryotic initiation factors, combines the 40s and 60s subunits
What is miRNA?
21-24 nt noncoding RNA guides that bind to mRNA and regulate mRNA abundance; 60% of genes of miRNA targeting sites; 1900 are present in the human genome, each can target multiple mRNAs
What is pre-miRNA?
product after the pri-miRNA is processed by the Drosha complex’s removal of the hairpin
How is the precursor to miRNA processed?
- Introns are removed from the primary miRNA transcript.
- 5’ cap and the poly-A tail are removed, creating the pri-miRNA.
- The Drosha complex removes the hairpin, creating pre-miRNA.
- The pre-miRNA is transported out of the nucleus and into the cytoplasm.
- The Dicer removes the terminal portion, creating the miRNA duplex.
- The miRNA duplex is degraded by the functional miRISC, creating an miRNA guide.
What does miRISC do?
melts away a strand in the miRNA duplex, leaving just 1 strand, the guide
What is a ribosomopathy?
phenotype that results from ribosomal mutations
What is an upstream open reading frame (ORF)?
uORFs are found in the 5’ UTR; they reduce translational output for mRNAs that encode very potent proteins by distracting ribosomes before they find initiation sequences
What is the reporter gene?
a gene that encodes a protein that is expressed when certain conditions are met; easily monitored experimentally, used to study the expression patterns of a target gene or the localization of its protein product; fluorescent reporter genes were used to study uORFs in the 5’ UTR in PTCH
What are RPL/RPS genes?
ribosomal protein large subunit genes and ribosomal protein small subunit genes; must have a balance in number to produce enough 80s ribosomes
RPS19, RPS24, and RPS17 mutations can decrease the amount of 40s subunits.
RPL5, RPL11, and RPL35A can decrease the amount of 60s subunits.
What is the 40S subunit?
small subunit part of the ribosome that binds to the 5’ methylated cap on mRNA, then migrates through the 5’ UTR looking for the initiation site
What is the 60s subunit?
large subunit part of the ribosome that catalyzes peptide bond formation via peptidyl transferase
What combines the 40s and the 60s subunits?
eukaryotic initiation factors (eIF)
What is the 80s ribosome?
eukaryotic ribosome, comprised of the 40s and 60s subunits combined; mature ribosome that can begin translation
List the five functions of an EJC.
- recruit other proteins to RNA
- influence the remaining splicing
- facilitate the export from the nucleus
- promote initial translation
- destroy mutant transcripts with premature stop codons (NMD)
Where are exon junctional complexes located?
placed 24 nts upstream of every exon-exon junction (where exons are spliced together)
How many proteins are present in an EJC?
4
Where are EJCs located in relation to
24
When do EJCs join RNA?
when splicing occurs
Are there EJCs within the open reading frame?
yes
Are there EJCs in the 3’UTR?
no
What is nonsense mutation decay (NMD)? When does it occur? What are the potential consequences?
when a stop codon is introduced prematurely (in addition to the normally placed one), causing ribosomes to stop translation early; this creates a truncated protein
What is no-go decay?
when there’s a problem with RNA that prevents ribosomes from translating
What is nonstop decay?
when a stop codon is not present in the mRNA, causing ribosomes to continue translating, leading to a series of incorrect amino acids
Why are truncated proteins unfavorable?
They are unable to interact effectively with other proteins because they lack a domain on the carboxy end
Where do tRNAs operate?
in the cytoplasm
What must happen before tRNAs can serve as adaptors between mRNA codons and amino acids?
must be charged by amino-acyl-tRNA synthetases, which covalently attach amino acids to form amino-acyl-tRNAs
Do anticodons only represent one codon?
No, they can be modified to recognize more than one.
How many common amino acids are there?
20
Where is the wobble position and what does it do?
Present at the third base in a codon; accommodates degeneracy with promiscuous pairing of standard and modified bases; this allows a cell to not need 61 nucleotides to be able to create 61 amino acids
Because of wobble, which is more prevalent, RNA or proteins?
There are more kinds of proteins than there is RNA codons.
What part of the ribosome is associated with the P site?
the peptidyl transferase
Where does the charged tRNA enter the ribosome?
the A site
Where does the charged tRNA exit the ribosome?
the E site
Where are ribosomes assembled?
in the nucleolus
What other things do ribosomes depend on?
nucleolus (for ribosomal production), RNA pol I and II (for rRNA transcription) and mRNAs transcribed by RNA pol II (for ribosomal proteins)
What does degeneracy mean?
In most cases, two or more nucleotide triplets specify a single one of the 20 amino acids
What is the function of the anticodon in tRNA?
It is available to base pair with its complement in mRNA.This interaction determines where an amino acid will be placed in a peptide chain.
Where is the wobble position?
the 5’ end of the anticodon
What direction does the ribosome move along the transcript?
5’ -> 3’
What are two translation regulation tools?
nuclease degradation and miRNAs
How does nuclease degradation work?
a shortened poly-A tail allows mRNA to be degraded by ribonucleases (especially polyA nuclease)
What is the consequence of a shortened poly-A tail?
less PABPC, less translation, more vulnerable to nuclease attack
What dictates the peptide sequence, ORF or bound proteins in ribonucleoprotein complexes?
ORFs
What dictates mRNA’s nuclear export, ORF or bound proteins in ribonucleoprotein complexes?
bound proteins in ribonucleoprotein complexes
What dictates mRNA’s localization in the cytoplasm, ORF or bound proteins in ribonucleoprotein complexes?
bound proteins in ribonucleoprotein complexes
What dictates mRNA’s engagement with translational machinery, ORF or bound proteins in ribonucleoprotein complexes?
bound proteins in ribonucleoprotein complexes
What dictates mRNA’s rate of degradation, ORF or bound proteins in ribonucleoprotein complexes?
bound proteins in ribonucleoprotein complexes
What do ribosomes normally do when they encounter EJCs? What happens to the EJCs after?
ribosomes reject EJCs and they’re recycled
What do ribosomes do to EJCs when they encounter a premature stop codon? What happens to the EJCs after?
They knock EJCs off and continue translating until they reach the stop codon, then they dissociate. The remaining downstream EJCs summon other proteins to degrade the RNA and virtually no effective protein is made.
Why doesn’t non-sense mediated decay (NMD) occur with single exon genes?
No splicing occurs, so there are no EJCs, so there are no proteins that summon other proteins for degradation.
