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- • sites of polypeptide assembly
- • eukaryotic ribosomes consist of 2 subunits of unequal size (60S & 40S)
- • are either found free in the cytoplasm or associated with the endoplasmic reticulum
- • differences between prokaryotic & eukaryotic ribosomes allow antibiotics to target ONLY bacterial not human ribosomes
What is the fate of proteins synthesized by ribosomes on the endoplasmic reticulum?
they are either exported from the cell or become part of the cellular membrane
The 60S ribosomal subunit contains which rRNAs?
The 40S ribosomal subunit contains what rRNA?
- regions flanking the coding region in mRNA
- • serve to stabilize the coding region
- • UTRs are why translation doesn’t start & stop at the ends of the mRNA
- • translation starts/stops internally because of these UTRs
- • the half life of mRNA is built into the sequence of the UTRs
Features of the Genetic Code
- 1. 4 bases in DNA, 20 AAs found in proteins
- 2. groups of 3 bases are called codons
- 3. for most AAs, there is more than 1 code word (the code is degenerate)
- 4. the code is nearly universal
Why is the genetic code called degenerate?
Because most of the 20 amino acids are coded for by more than one codon
a trinucleotide sequence of mRNA that determines which amino acid is added to a nascent protein during translation
Start Codon Sequence
codes for Methionine
- • tRNAs link correct AAs based on what’s CODON-ed for in the mRNAs
- • each amino acid binds to 1 or several specific tRNA molecules
- • all tRNAs contain ~80 nucleotides
- • have a characteristic cloverleaf due to residue base pairing
- • called charged when linked up with an AA
What is the purpose of CCA on the tRNA?
- • the final Adenosine of CCA is the site of attachment of the amino acid to the tRNA
- • every tRNA has a CCA sequence at its 3' end added after synthesis by RNA pol III
Why attach an amino acid to the 3' of tRNA?
- • formation of a peptide bond between the amine group of one amino acid & the carboxyl group of another is thermodynamically unfavorable
- • energy barrier is overcome by ACTIVATING amino acids
- • this is accomplished by linking them via their hydroxyl group on a ribose unit at the 3' end of tRNA (high energy linkage)
- enzymes that activate & attach amino acids to their respective tRNAs
- • they pair the correct amino acid with its corresponding tRNA
- • there's a different synthetase for EVERY AA
- • there are fewer tRNAs than there are codons
- • the 3' end of the codon allows for a more relaxed bonding with the 5' base of the anticodon
- • the potential for non-Watson-Crick base pairing at this position allows a single tRNA to pair with several different codons
What is the starting point for translation?
- ribosomal SUBUNITS
- NOT fully assembled ribosomes
In which direction does a ribosome read RNA?
- 5' to 3'
- (same direction as nucleic acid synthesis by DNA & RNA pols)
- • an initial step: binding of the initiator Met-tRNA to eIF2 & GTP
- • this complex then binds to the 40S ribosomal subunit
- • with the help of more factors (eg. eIF4), the 40S complex associates with mRNA
- • the 40S complex (+ additional factors) migrate in the 5’ to 3’ direction searching for the first AUG initiation codon
- • upon localizing an initiation codon, eIF2 is released
- • this permits addition of the 60S subunit
functions in the initiation process
• unlike all other AA-tRNAs, it can bind to the small ribosomal subunit (40S) with the help of eIF-2 & the binding is NOT dependent on the presence of a codon for methionine
binds to initiator Met-tRNA & GTP (eIF2-GTP-Met-tRNA) to bind to the small 40S ribosomal subunit & initiate translation
• once the anticodon of the initiator Met-tRNA & the AUG start codon match, the eIF-2 allows hydrolysis of GTP that enables the release of eIF-2 from the 40S subunit & allows the 60S ribosomal subunit to join
recognized by translational factors (helps identify it’s not junk RNA but that it’s meant to be TRANSLATED) who say this is a substrate for translation
recognizes + associates with the 5' m7G cap and positions the mRNA on the small 40S ribosomal subunit
Which subunit first binds the the 5' end of a mRNA?
What are the 3 sites that interact with tRNAs during elongation of a peptide?
A, P & E sites
• A site accommodates the incoming amino acyl-tRNA
• P site contains the peptidyl-tRNA complex (the tRNA still linked to all the amino acids added to the chain so far)
• E (exit) site is for empty tRNA that has been used
~ GTP hydrolysis provides energy for the translocation step ~
a covalent bond between an amino group of 1 molecule & the carboxyl group of another; are formed between amino acids during protein synthesis
• catalyzes peptide bond formation (the nucleophilic attack by the amino group of the aminoacyl-tRNA in the A site on the carbonyl of the activated ester linkage of the peptidyl-tRNA in the P site)
• as a result, growing polypeptide chain is transferred to the tRNA in the A site
• a component of the 28S rRNA (a ribozyme)
Formation of each peptide bond requires considerable:
• 2 high-energy bonds are used each cycle (EF-1 and EF-2)
• two high-energy phosphates are used to generate each aminoacyl-tRNA
Elongation Factors (EFs)
position amino acyl-tRNAs on the ribosome & promote RNA translocation
- • "chaperones" a tRNA into the A site
- • catalyzes the GTP-dependent binding of aminoacyl-tRNA to ribosomes, thereby regulating the fidelity & rate of polypeptide elongation during translation
- • an abundant protein in eukaryotes
translocates tRNA from the A (1st) site to the P (2nd) site, making room for new tRNA to bind & attach another amino acid
Where is the termination codon located in the mRNA?
NOT AT THE 3' END AMIRITE?!
Termination (Release) Factor
- • binds to the A site & promotes the cleavage of the polypeptide chain from the tRNA in the P site
- • there are no tRNAs with anti-codons complementary to the stop codon sequences
- • elongation stops when one of these triplet stop codons appears opposite the A site on the ribosome
- • this is followed by the release of the ribosome from the mRNA
mRNA that will code for only 1 polypeptide sequence
• with very few exceptions, eukaryotic mRNA is monocistronic
• bacteria, on the other hand, frequently have polycistronic messages
multiple initiation events that usually take place on each mRNA being translated; ribosomes can be spaced as close as 80 nucleotides apart along a single mRNA
a single base substitution in which 1 amino acid replaces another
• can activate cryptic splice sites, meaning a mutation can set up an exon that’s supposed to be an intron
a change in a codon that converts it to a stop codon, leading to premature termination of translation
Effects of base substitutions on translation:
- • cryptic splice site generation (can create or remove an exon)
- • generation of termination signal
- • loss of termination signal
- • frame-shift mutations: can change the entire mRNA sequence & how it's supposed to be read
causes the codons to be read out of frame, thereby leading to a misinterpretation of mRNA
What happens during periods of stress to regulate translation?
- • phosphorylation of eIF2
- • phosphorylated eIF2 is inactive, thereby conserving energy by halting protein synthesis
- • play an important role in the regulating translation
- • are about 21 nt long, non-coding & usually target the 3’ UTR
- • they recruit the RISC complex & either cause stalling of the translation complex or degradation of mRNA being translated
- • if the miRNA is a perfect complimentary match to the 3’ UTR it can cause degradation
- • if it’s not a perfect match it just blocks the translational machinery
IRES (Internal Ribosome Entry Sites)
- specialized sequences in viruses that can recruit the pre-initiation complex in a manner that doesn’t involve the initial scanning process
- • viruses can come in & take over the whole translation process
- • have this way of assembling eukaryotic ribosomes on the IRES sequence
What is protein synthesis very important for in relation to the CNS?
learning & memory
True or False: Interaction of the eIF2-GTP-Met-tRNA complex with the 40S ribosomal subunit must occur in the presence of mRNA?
False, It typically occurs in the absence of mRNA
What is the function of eIF2 during translation?
It binds the 40S subunit to tRNA
What happens to eIF2 when the anticodon of the initiator tRNA matches the AUG start codon?
GTP is hydrolyzed, allowing eIF2 to dissociate from the ribosome and the 60S subunit to bind to the 40S subunit
Which elongation factor is required for tRNA to bind to the A site of the ribosome?
Which elongation factor is required for translocation of tRNA from the A site to the P site of the ribosome?
Which enzyme is thought to catalyze the nucleophilic attack on the carbonyl group of the peptidyl-tRNA by the amino group of the aminoacyl-tRNA? Where does this reaction occur on the ribosome?
The enzyme is peptidyl transferase and it occurs in the A site of the ribosome
What is the function of the soluble protein called termination factor?
It promotes the cleavage of the polypeptide chain from the tRNA in the P site, releasing the mRNA from the ribosome
In bacteria, the region of the 3' terminus of the 16S ribosomal RNA is complementary to the _______ (which sequence)
Shine-Dalgarno Sequence; This is the ribosome binding site on the mRNA. In eukaryotes the equivalent sequence is known as the Kozak sequence.