Protein Synthesis

Learning objectives: understand the following concepts

Revisiting:

• The genetic code is ___ (non-overlapping).

It is read continuously with no ___.

• There are 3 possible reading frames on mRNA, but only ___ is correct.

Revisiting the Wobble Hypothesis

Of the possible 64 triplet codons, 61 code for amino acids and 3 serve as _ codons.

__-accepting tRNAs - different tRNAs that accept the same amino acid. Note that aminoacyl-tRNA-synthetases are responsible for specific conjugation of an AA to cognate tRNA.

Many tRNAs can bind to two or three codons that specify the same amino acid due to the presence of modified bases in the __ (__) position in their anticodon.

One example is yeast tRNA-Phe that has the anticodon 5'—GmAA—3', which recognizes the codons 5'—UUC—3' and 5'—UUU—3'.

It is, therefore, possible for non-Watson-Crick base pairing to occur at the third position of the codon. This phenomenon is called the ___ ___.

Top: Diagram showing nucleotides of tRNAs that are found in the wobble position (5') in the anticodon (blue) and the various nucleotides at the 3' position of the codon (red) that can be found in non-Watson-Crick base-pairs.

Bottom: Diagram showing nucleotides found in the 3' position of the codon (blue), and the 5' wobble position of the anticodon (red).

Revisiting acceptor and anticodon arms of tRNA

Acceptor arm contains __, which is present in all tRNAs

The ___ arm is important for interpreting the coding sequence in
mRNA during translation by ribosomes.

The first codon codes for ____

In prokaryotes, N-formylmethionine is the first AA

In eukaryotes, ___ methionine is used

The process of protein synthesis

1. Synthesis proceeds from the _-terminus to the _-terminus of the protein.

2. The ribosomes "read" the mRNA in the _ to _ direction.

3. Active translation occurs on polyribosomes (also termed ___). This means that more than one ___ can be bound to and translate a given mRNA at any one time.

4. Chain ___ occurs by the sequential addition of amino acids to the C-terminal end of the ribosome-bound polypeptide.

5. When a stop codon is reached, the ribosome ___ synthesis.

Ribosome binding site at 5' end of mRNA

Note: In prokaryotes, the purine-rich region upstream of the initiation codon, known as the __-___ sequence (red), is important for locating the INITIATION codon (blue).

It is found at the __ side of each initiator
AUG codon in prokaryotic mRNAs.

This sequence is complementary to
sequences present near the 3'-end of the __ rRNA.

In prokaryotes, the Shine-Dalgarno sequence on mRNA base pairs with
3'-end of 16S rRNA to accurately position the initiation codon on SSU.

In eukaryotes, initiator AUG codons are generally, but not always, the ___ encountered by the translational machinery.

A specific sequence context surrounding the initiator AUG aids ribosomal discrimination. This context is
A/GCCA/GCCAUGA/G in most mRNAs and is referred to as the __ ___ ___.

Protein synthesis - translating mRNA to protein

Steps of translation initiation:

• Binding of tRNA-Met to SSU aided by ___

• Recruitment of mRNA to SSU
aided by __ & __

• Poly A binding protein (PABP, green) mediates the formation of a ___-___ by simultaneously binding to poly-A and elF4G.

• Note that elF2 is a GTP binding protein that forms a ___ complex with Met-tRNA

• __ and ___ recognize the
5' end cap of mRNA and recruit it
to SSU.

• ___ mediates the formation of a
closed-loop by simultaneously
binding to poly-A and elF4G

More steps of translation initiation

1. Binding of tRNA-Met to AUG through base pairing, allowing correct insertion of to the __ site.

2. GTP hydrolysis induces the release of ___ and LSU binds to SSU.

3. Binding of a second aminoacyl-tRNA to the __ site aided by EF-Tu

4. Formation of the ___ bond and release of EF-Tu.

5. Peptide bond formation is catalyzed by __ __, a ___ in the LSU

Note: one ATP and two GTP are used for making one peptide bond (2 GTPs here and 1 ATP in aa activation). Also, tRNA-Met is free of aa at this step.

elF2: eukaryotic initiation factor 2

EF-Tu: elongation factor Tu

Mechanism of peptide bond formation

Nucleophilic attack on carboxylate of
the aa in __ site by the amino group of
the aa in the __ site.

Peptide bond formation leading to
_____ of tRNA in the P site

Peptide bond formation is catalyzed by
___ ___ — a ribozyme in the LSU

Summary slide for initiation and elongation

1. tRNA-Met occupies the first codon at the _ site

2. A new aminoacyl-tRNA aided by EF-Tu-GTP binds in the _ site

3. EF-Tu release and peptide bond ___

4. tRNA in __ site becomes aa free

5. EF-G-GTP aids the ribosome to move to 3' direction on the mRNA and aa free tRNA moves to the _ site

6. The tRNA in the E site exits and the process continues until a __ codon (UAA, UAG or UGA) is encountered at the _ site.

Termination of protein synthesis

Translation termination occurs when a
__ codon is encountered within the
context of the _-site of the 60S subunit.

The termination factor __, which binds
GTP, then stimulates the peptidyl-
transferase ribozyme to transfer the
peptide, from the tRNA in the P-site to
H2O coupled with the hydrolysis of GTP.

The peptide is ___ from the ribosome along with the naked tRNA and eRF-GDP complex.

The anti-association factors then promote dissociation of the two ribosomal subunits and the process can begin ___

Multiple ribosomes translate a single mRNA

The antibiotic puromycin ___ protein synthesis

Puromycin can be inserted into the _ site and make a covalent bond leading
to ___ of protein synthesis.

Note that puromycin resembles the
___ nucleotide in the 3' end of the tRNA.