Chapter 15 Flashcards
mRNA open reading frame ORF
Open reading frame (ORF): continuous, non-overlapping string of codons (3 nucleotides)
in the protein-coding region of an mRNA.
Translation starts at the 5’end of the ORF and proceeds one codon at a time to the 3’ end.
In bacteria, the start codon is usually 5’-AUG-3’, but 5’-GUG-3’ and sometimes 5’-UUG-3’ are also used. Eukaryotic cells always use 5’-AUG-3’ as start codon. Stop codon: UAA, UAG and UGA.
mRNA recruitment of tanslation machinery
Prokaryotic mRNAs have a ribosome-binding site that recruits the translational machinery
• RBS (ribosome-binding site): short sequence upstream of the start codon. Also referred to as a Shine-Dalgarno sequence.
• The RBS, located 3-9bp on the 5’ side of the start codon (seq: 5’- AGGAGG-3’), is complementary to a sequence located near the 3’ end of one of the ribosomal RNA (16s rRNA). The core of the region the 16s rRNA has the sequence 5’-CCUCCU-3’. The extent of complementarity and spacing between the RBS and the start codon has a strong effect on how actively a particular ORF is translated. High complementarity and proper spacing favors translation.
• Some prokaryotic ORFs, not first ORFs, lack a strong RBS, but are nonetheless actively translated. In these cases, the ORFs usually overlaps. 5’-AUGA-3’ (contains a start codon and stop codon). Called translational coupling. In this situation, the translation of the downstream ORF requires translation of the upstream ORF.
17-6
tRNA overview/structure
Transfer RNAs are adaptors between codons and amino acids
• There are many types of tRNA molecules, but each is attached to a specific amino acid, and each recognizes a particular codon or codons.
• tRNA molecules are between 75 and 95 ribonucleotides in length.
• All tRNAs have the 3’ terminal sequence 5’-CCA-3’ (CCA adding
enzyme: synthesize RNA without a template).
• Presence of several unusual bases in their primary structure. (pseudouridine (ψU), dihydrouridine (D), hypoxanthine). This leads to improved tRNA function. Cells lacking these modifications show reduced rate of growth.
tRNA secondary structure
Resembled a clover leaf. It has single-stranded and Double-stranded regions.
A. Acceptor stem (arm): attach amino acid. double strand, and 3-end protrusion.
B. ψU loop. 5’-TψUCG-3’
C. D loop: has dihydrouridine.
D. anti-codon loop: contains the anticodon,
a three nt long sequence that is responsible for
recognizing the codon by base pairing with
the mRNA.
E. Variable loop. 3-21 bases
tRNA charging steps
Two steps of tRNA charging: attachment of amino acid to tRNA
• Charging requires an acyl linkage between the carboxyl group of the amino acid and the 2’ or 3’ hydroxyl group of the adenosine nucleotide that protrude from the acceptor stem at the 3’ end of the tRNA. (High energy bond, release energy during hydrxolysis. Significant for protein synthesis).
tRNA charging:
tRNA synthetases
Class I enzyme: attaches the amino
acid to the 2’-OH of the tRNA. (monomeric)
Class II enzyme: attaches the amino
acid to the 3’-OH of the tRNA. (dimeric or tetrameric)
Mech of aminoacyl-tRNA synthetase
Each aminoacyl-tRNA synthetase attaches a single amino acid to one or more tRNAs. The same tRNA synthetase is responsible for charging all tRNAs for a particular amino acid.
Most organisms have 20 different tRNA synthetases, but this is not always the case. Some
bacteria lack the tRNA synthetase for Glutamine. The use tRNA synthetase for glutamate, and 17-13 a second enzyme to convert tRNA (glu) to tRNA (gln).
How to ensure accuracy regarding aminoacyl-RNA synthetase
tRNA synthetases recognize unique structural features of cognate tRNAs
• The challenge for aminoacyl-tRNA synthetases: (A) they must recognize the correct ste of tRNAs for a particular amino acid, and (B) they must charge all these iso- accepting tRNAs with correct amnio acid.
• The specificity determinants are clustered at two distance sites on the molecule: the acceptor stem and the anticodon loop.
• In some cases, the discriminator base in the acceptor stem can determine the specificity of tRNA from one synthetase to another. (by mutation study)
• Serine is specified by 6 codons, so the synthetase for serine must rely on determinants that lie outside of anti-codon.
Aminoacyl-tRNA formation accuracy
Aminoacyl-tRNA formation is very accurate
• Selecting the right amino acid is more challenging than recognizing the appropriate tRNA.
• Frequency of misincorporation:
<0.1%.
Some amino acids (like cysteine and tryptophan) differ substantiallyin size, shape and chemical groups.
(easy to distinguish)
Tyr can form hydrogen bond, phenylalanine cannot. Valyl-tRNA synthetase can sterically exclude isoleucine from
It catalytic pocket because isoleucine is larger than valine.
But valine should slip easily into the isoleucyl-tRNA synthetase amino-acid binding site. The extra mythelene group provide
-2 to 03 kcarl/mole of energy. This small difference In free energy will make binding to isoleucine ~100-fold more likely than binding to valine.
How is this additional level of fidelity achieved?
Aminoacyl-tRNA proofreading mech
Some aminoacyl-tRNA synthetase use an editing pocket to charge tRNA with high accuracy
• Proofreading to increase fidelity.
• For example, in addition to its catalytic pocket (for adenylylation), isoleucyl tRNA synthetase has a nearby editing pocket that allows it to proofread the product of adenylylation. AMP-Valine can fit into the editing pocket, where it is hydrolyzed and released as free valine and AMP.
• So, two discrimination steps: one is the initial binding and adenylylation of the amino acid (discrimination factor of ~100), the other is the editing of the adenylylated amino acid (discrimination factor of ~100). Overall, less than ~0.01% error rate.
Ribosome discrimination between correctly and incorrectly charged tRNAs
The ribosome is unable to discriminate between correctly and incorrectly charged tRNAs
• Genetic and biochemical evidence
1. Mutation in the anticodon. tRNA synthetases frequently don’t rely on interaction with the anti-codon to recognize the cognant tRNA. Hence, a subset of tRNA can be mutated in their anticodons but still be charged with their usual cognate amino acid. However, the mutated tRNA delivers amino acid to the wrong codon. That means ribosome does little to prevent an incorrectly charged tRNA from adding an inapproproate amino acid to the growing polypeptide.
2. Biochemical experiment nicely illustrates the point that ribosome recognizes tRNA and not the amino acid that it is carrying.
Ribosome
The ribosome: a macromolecular machine that directs the synthesis of proteins
Ribosome: at least 3 RNA molecules, and more than 50 different proteins, speed: only 2-20 amino acid per second.
In prokaroytes, the transcription machinery and the translation machinery are located in the same compartment. Thus the ribosome can commence translation of the mRNA as it emerges from the RNA Polymerase.
In eukaryotes, transcription is in the nucleus, whereas translation is in
the cytoplasm.
Ribosomal subunits
The large subunit: the peptidyl transferase center, which is responsible for the formation of peptie bonds.
• The small subunit: decoding center, in which charged tRNAs read or decode the codon units of the mRNA.
• Size: bacteria 30S and 50S; eukaryotic ribosome: 40S and 60S.
• Based on the velocity of their sedimentation after aultracentrifugation. S: Sverdberg (inventor of ultracentrifuge)
The large and small subunits undergo association and dissociation during each cycle of translation (The ribosome cycle)
Binding of the mRNA and initiator to the small ribosome subunit
Recruits a large subunit Protein synthesis (initiation)
Ribosome translocation from one codon to the next. (translation elongation)
Complete polypeptide release and ribosome dissociation
Poly some
Although a ribosome can synthesize only one polypeptide at a time, each mRNA can be translated simultaneously by multiple ribosomes. A mRNA bearing multiple ribosomes are known as a polyribosome or a polysome. A 1000-base ORF can bind to more than 10 ribosomes.
The peptidyl transferase reaction
The peptidyl transferase reaction
• Two substrates: an aminoacyl-tRNA and a peptidyl-tRNA.
• The aminoacyl-tRNA is attached at its 3’ end to the carboxyl group of the amino acid.
• The reaction to form a new peptide bond is called
the peptidyl transferase reaction.
So the bond between the aminoacyl-tRNA and the amino acid is not broken during the formation of the next peptide bond. Instead, the bond between the peptide-tRNA and the growing peptide is broken. This reaction takes place without the simultaneous hydrolysis of a nucleotide triphosphate. Why? High energy acyl bond from the charging reaction. One ATP is needed for each tRNA charging reaction.
Ribosome binding sites for tRNA
The ribosome has three binding sites for tRNA
• To perform the peptidyl transferase reaction, the ribosome must be able to bind at least two tRNAs simultaneously.
• A-site: the binding site for aminoacylated-tRNA
• P-site: the binding site for peptidyl-tRNA
• E-site: the binding site for the tRNA that is released after the growing peptide chain has been transferred to the aminoacyl-tRNA (E is for “exiting”).
Each tRNA-binding site is formed at the interface between the large and the small subunits of the ribosome.
• In this ways the bound tRNA can span the distance between the peptidyl transferase center in the large subunit and the decoding center in the small subunit.
• The 3’ ends of the tRNAs that are coupled to the amino acid or to the growing peptide chain are adjacent to the large subunit. The anticodon loops of the bound tRNAs are located adjacent to the small subunits
Factors involved during the initiation of translation
For translation to be successfully initiated:
The ribosome must be recruited to the mRNA.
A charged tRNA must be placed into the P-site of the ribosome.
The ribosome must be precisely positioned
over the start codon. (critical for the reading frame)
Prokaryotic mRNAs are initially recruited to the small subunits by base pairing to rRNA
For ideally positioned RBSs, The small subunit is positioned on the mRNA such that the start codon will be in the P-site when the large subunit joins the complex.
Translation initiation mechanism involving tRNA
A specialized tRNA charged with a modified methionine binds directly to the prokaryotic small subunit
Translation initiation is the only time a tRNA is binding to the P-site without previously occupying the A-site. The event requires a special tRNA (initiator tRNA) , which base pairs with the start codon: usually AUG or GUG.
AUG and GUG have a different meaning when they occur within an ORF, where they are read by tRNA for methionine and valine respectively.
Although the initiator tRNA is first charged with a methionine, a formyl group is rapidly added to the methionine amino group by a separate enzyme (Met-tRNA transformylase). Thus rather than valine or methioine, the initator tRNA is coupled to N-formyl methionine. The charged tRNA is refered to fMet- tRNAfMet.
In prokayotes, a enzyme known as deformylase removes the formyl group form the amino terminus during or after the synthesis of the polypeptide chain. In fact, many mature prokayotic protein do even start with methionine; aminopeptidases often remove the amino-terminal methionine as well as one-two additional amino acid
Three initiation factors direct the assembly of an initiation complex that contains mRNA and the initiator tRNA
Translation initiation factors: IF1, IF2 and IF3
IF1: prevent tRNAs from binding to the portion of the
small subunit that will be part of the A-site. (binds to the A-site) IF2: is a GTPase that interacts with three key components of the intiation machinery: the small subunit, IF1 and the charged tRNA. It facilitates the association of fMet-tRNA with the small subunit and prevents other charged tRNAs from associating with the small subunit.
IF3: binds to the small subunit and blocks it from re-associating with large subunit. (occupy the future E-site)
With the binding of three initiation factors, the small subunit is
preparing to bind to the mRNA and the initiator tRNA.
The last step of initiation involves the association with the large
subunit to create a 70S initiation complex. In this step, IF3 is released, and the large subunit is free to bind to the small subunit. This interaction stimulates the GTPase activity of IF2•GTP. IF2•GDP has reduced affinity for the ribosome and the initiator tRNA, leading to the release
of IF2•GDP as well as the IF1 from the ribosome.
Prokaryotic initiation and eukaryotic initiation comparisons and contrasts
Prokaryotic initiation and eukaryotic initiation
Similarity:
Both use a start codon, and a dedicated initiator tRNA, both use initiator factors to form complex with the small ribosomal subunit that assembles on the mRNA before addition of the large subunit.
Difference:
The small subunit is already associated with an initiator tRNA when it is recruited to
the capped 5’ end of the mRNA.
The small subunit scans along the mRNA in a 5’ to 3’ direction until it reaches the first 5’-AUG-3’. (explains why the vast majority of eukaryotic RNAs encode a single polypeptide.
More auxiliary factors are needed.
PolyAntail in translation initiation
Translation Initiation factors hold eukaryotic mRNAs in circles.
The presence of a Poly-A tail contributes to the efficiency of
eukaryotic translation.
Mediated by eIFG binding directly to the 3’ end of mRNA and the Poly-A binding protein.
Result in the circular configuration of the mRNA, several rounds of translation.
uORF
Sometimes the first ORF is in a poor sequence context, resulting in its frequent bypass.
• In other cases, short upstream ORFs (uORFs, less than 10 AAs) are translated. This allows interaction between initiation factors (eg. eIFG and eIF3) that tether the 40S subunit to the mRNA to be retained after termination. They can scan the next AUG after binding to a new initator tRNA complex.
IRES: internal ribosome entry site
IRES: internal ribosome entry site
• A more example of initiation at sites downstream from the most 5’-proximal AUG are internal ribosomal entry sites (IRES). IRESes are RNA sequences that function like prokayoritc RBSs. The recrut the small subunit to bind and initiate even in the absence of a 5’ cap. The are often encoded in viral mRNAs that lack a 5’ cap and have a need to exploit the sequence of their genome maximally.
IRES mechs
Two mechanisms: 1. Recruit eIFG
2. Cricket paravirus virus
The 5’-UTR of the mRNA forms
a complex RNA structure that mimics a tRNA bound to the P
-site of the 40S ribosomal subunits. This way it bypass all the initiation factors and an initiation tRNAs.
This leads to the hypothesis that early in evolution all mRNAs have such IRESs and that initiation factors evolved later to make translation more efficient and versatile.
