L12: Translation Flashcards
define translation
the order of nucleotides in messenger RNA (mRNA) is converted into the linear sequence of amino acids in a protein
what is messenger RNA (mRNA)
information contained in the open-reading frame (ORF)
mRNA - what is the open reading frame (ORF)
a contiguous, non-overlapping string of codons that specifies protein sequence
Open reading frame (ORF) - what are the start and stop codons
- Start codons: AUG
- stop codons: UAA, UAG, UGA
what is transfer RNA (tRNA)
- an adapter molecule between codons and amino acids
- 3’ terminus has a 5’-CCA-3’ sequence that binds cognate amino acids
- developed by Crick’s Adaptor Hypothesis
tRNA secondary structure
- resembles a clover leaf:
1. acceptor stem
2. anticodon loop
tRNA secondary structure - acceptor steam
attachment site for amino acid
tRNA secondary structure - anticodon loop
contains anticodon which base pairs with the codon
tRNA tertiary structure
- “L” shape
- is called aminoacyl-tRNA when the RNA is “charged”
tRNA tertiary structure - “charging” of tRNAs
- amino acid is attached to 3’ terminal A nucleotide of tRNA
- the linkage is a high energy bond between tRNA and amino acid - yields energy when cleaved
tRNA tertiary structure: “charging” of tRNAs - aminoacyl-tRNA synthetase
- enzyme that charges tRNA
- each amino acid has one dedicated aminoacyl-tRNA synthetase
what are ribosomes
- it is what makes the polypeptide sequence from mRNA
- its is comprised of both proteins and RNA
- consists of 2 subunits:
1. large subunit
2. small subunit
ribosomes - why is it important for eukaryotes to separate transcription and translation
RNA processing and splicing needs to occur after transcription
what are ribosomes - large subunit
contains peptidyl transferase center that forms peptide bonds between amino acids
ribosomes: large subunit - how is the peptide bond created
- dehydration reaction - water is kicked out
- bond is formed between the carboxyl and amino group
ribosomes - small subunit
contains decoding center where mRNA codons are read by charged tRNAs
ribosomes - eukaryotes vs prokaryotes
- prokaryotes are smaller and eukaryotes are larger
- but there are individual ribosomes with differing sizes
rRNA processing
- rRNA begins as a single transcript (pre-rRNA) that is cleaved
- done so cleaved parts can be incorporated into the ribosome
explain the ribosome cycle
- ribosome subunits assemble on RNA
- mRNA is translated into a polypeptide
- ribosome dissociates
ribosome cycle - what is a polysome
- each mRNA can be translated by more than one ribosome simultaneously
- so a transcript can have many ribosomes on it
what direction is a polypeptide synthesized in?
- amino-to carboxy direction
- each new amino acid is added to the carboxyl terminus of a growing polypeptide chain
explain peptide bond formation
- called a peptidyl transferase reaction
- involves:
1. peptidyl-tRNA
2. aminoacyl-tRNA
peptide bond formation - peptidyl-tRNA
- attached to a growing polypeptide chain
- the one already in ribosome
peptide bond formation - aminoacyl-tRNA
- the incoming charged tRNA
- the high energy bond is not broken during the formation of the new peptide bond
peptide bond formation - if the high energy bond in aminoacyl-tRNA is not being cleaved, which tRNA is being claved?
- peptidyl-tRNA
- the high energy bond between the peptidyl-tRNA and polypeptide chain is broken
- the released energy drives the formation of a new peptide bond
what are the steps of translation?
- initiation
- elongation
- termination
initiation of translation - explain the ribosome tRNA binding sites
- A-site
- P-site
- E-site
initiation of translation: ribosome binding site - A-site
binding site for aminoacyl-tRNA
initiation of translation: ribosome binding site - P-site
binding site for peptidyl-site
initiation of translation: ribosome binding site - E-site
“exiting” site for tRNA released after the peptidyl transferase reaction
initiation of translation - what are the three requirements for initiation?
- ribosome is recruited to the mRNA
- the charged tRNA is placed at the P-site
- the ribosome is precisely positioned over the start codon to establish the reading frame
initiation of translation - prokaryotic
- they contain a ribosome binding site (RBS) termed Shine-Dalgarno sequence
- this sequence is complementary to a part of the 16S rRNA in the small subunit.
- the 16S rRNA base pairs with the Shine-Dalgarno sequence
- this then places the start codon at the P-site
- the initiator tRNA (charged with methionine) binds in the P-site of the ribosome
initiation of translation - eukaryotes
- translation is initiated by multiple features:
1. 5’ cap recruits ribosome to transcript and scans for 1st 5’-AUG-3’
2. Kozak sequence surrounds AUG and enhances translation efficiency - the small subunit of the ribosome associates with the initiator tRNA before mRNA binding
initiation of translation: eukaryotes - Kozak sequence
- 5’ - G/A NN AUG G - 3’
- it interacts with the tRNA, not the ribosome
initiation of translation: eukaryotes - why is it important that the small subunit associates with the initiator tRNA before mRNA binding
- the complex is recruited to the 5’ cap of the mRNA and it scans in the 5’-3’ direction for the first AUG start codon
- this start codon is recognized by the initiator tRNA anti-codon
- once the start codon is recognized (via the initiator tRNA), the large subunit joins
translation elongation - three key events
- correct aminoacyl-tRNA is loaded into the A-site (dictated by the codon on mRNA)
- peptidyl transferase reaction occurs between the A-site and P-site polypeptide chain
- peptidyl-tRNA in the A-site moves to the P-site
translation elongation - prokaryotes vs eukaryotes
process is similar for both
translation elongation: prokaryotes - elongation factors (EFs)
- EF-Tu
- EF-G
prokaryotic translation elongation: elongation factors (EFs) - EF-Tu
- it guides the aminoacyl-tRNAs to the ribosome and complexes with GTP
- EF-Ru-GTP-aminoacyl-tRNA complex interacts with the factor-binding center of large ribosomal subunit
- GTP is then hydrolyzed and EF-Tu is released from tRNA and ribosome
prokaryotic translation elongation - what happens after EF-Tu is released?
a peptidyl transferase reaction occurs between the A-site amino acid and P-site polypeptide chain
prokaryotic translation elongation - after the peptidyl transferase reaction occurs, what must happen to the ribosome?
- translocation:
- the P-site tRNA must move to the E-site
- the A-site tRNA must move to the P-site
- and the mRNA must move by three nucleotides to expose the next codon
prokaryotic translation elongation: EF-G - how does it start off?
- required for translocation
1. binds to GTP and associates with the ribosome
2. it contacts the factor-binding center of the large subunit and GTP is hydrolyzed
prokaryotic translation elongation: EF-G - what happens after the GTP is hydrolyzed?
- EF-G undergoes conformational change and binds to the A-site
- tRNA in A-site is pushed into P-site, which forces the P-site tRNA into the E-site
prokaryotic translation elongation: EF-G - what happens after the tRNA is pushed to the E-site?
- base pairing between the tRNAs and mRNA is maintain, which moves the mRNA over by three nucleotides
- EF-G-GDP and tRNA in the E-site are released and the ribosome is ready for the next amino acid
termination of translation
- one of the three stop codons enters the A-site
- it is recognized by release factors (RFs) that free the polypeptide chain
- triggers hydrolysis of the polypeptide chain from the tRNA in the P-site
in prokaryotes: how is translation regulated?
- ribosomal proteins repress their own synthesis
- ribosomes consists of both RNA and protein
- you need an appropriate number of proteins and ribosomal RNA
in prokaryotes: translation regulation - what happens when rRNA levels are high
- ribosomal proteins bind to high-affinity binding sites of rRNA
- this promotes ribosomal assembly
in prokaryotes: translation regulation - what happens when all rRNA molecules are bound by ribosomal protein
- there is no need to make more protein
- bc there are no high-affinity binding sites, the ribosomal protein binds to low-affinity binding sites near the RBS
in prokaryotes: translation regulation - what happens when the ribosomal protein binds to low-affinity binding sites?
- this prevents translation initiation
- and prevents translation of a second ribosomal protein due to the transcript folding back (via intermolecular base pairing)
why is it important to have translational-dependent regulation of mRNA?
bc mRNA is susceptible to breaks due to it being single stranded
what is an example of translational-dependent regulation of aberrant mRNA?
stalled ribosomes on broken prokaryotic transcripts
stalled ribosomes on broken prokaryotic transcripts - how is this dealt with?
- the ribosome reaches the end of the broken transcript before encountering a stop codon
- but it doesn’t fall off, it is instead rescued by tRNA-mRNA chimera molecule (tmRNA) termed **SsrA*
stalled ribosomes on broken prokaryotic transcripts - what happens after tmRNA rescues the ribosome
- the tRNA-like 3’ structure is charged with Ala and assembles with EF-Tu-GTP
- it then binds to the A-site (only fits if the transcript is broken due to its large size)
- it then undergoes a peptidyl transferase reaction
stalled ribosomes on broken prokaryotic transcripts - what happens after the peptidyl transferase reaction?
- translocation releases the broken transcript
- the mRNA component of the tmRNA is translated
stalled ribosomes on broken prokaryotic transcripts - how does the ribosome dissociate?
- there are codes for 10 amino acid residues before reaching the stop codon which then allows for dissociation
- the 10 amino acids serve as a signal for the cell to degrade the product
- degradation prevents detrimental effects of truncated product to cell
