Ribosome & Protein Translation: Prokaryotic

Ribozyme

• the ribosome is a ribozyme predominantly composed of RNA, with its catalytic activity driven by ribosomal RNA (rRNA)

  • ribosome is a ribozyme that catalyzes biochemical reactions

• both tRNA (yellow) and mRNA are RNA molecules

• consequently, translation is primarily an RNA-driven process

Intro to Translation

• translation is the mRNA-guided process leading to protein synthesis by ribosomes, and is divided in 5 steps

• the ATP-dependent activation of amino acid mediated by the aminoacyl tRNA synthetase

• step 2-4 that are mediated by the ribosome

• last step is protein folding

Prokaryotic Translation & Polysomes

• the mRNA tends to be short-lived in prokaryotes and translation is therefore tightly coupled to transcription

• translation occurs on mRNA while they are still transcribed by RNA pol → forming polysomes

  • polysome = a cluster of ribosomes held together by a strand of messenger RNA that each ribosome is translating

• multiple ribosomes are loaded on the 5’ end of mRNA forming dense structures

  • following assembly of the first ribosome on the mRNA, it moves toward the 3′ end, freeing up the 5’ end and allowing new ribosome molecules to associate

• initiation is initiated near the 5’end of mRNA

Prokaryotic Translation & Polysomes FIGURE

Prokaryotic Ribosomes

• prokaryotic and eukaryotic ribosomes have similar architectures, with eukaryotes being larger

• prokaryotic ribosome is formed by the 30S and 50S subunits

  • the two subunits will assemble on top of each other, leaving a cleft region, which is where mRNA will be threaded thru

• it contains ~50 proteins and 3 major rRNA molecules: 5S and 23S in the large subunit and 16S in the small subunit

Assembled Prokaryotic Ribosome

• the assembled prokaryotic ribosome forms the 70s complex

  • there are 3 major binding sites for tRNA:

    • aminoacyl (A)

    • peptidyl (P0

    • exit (E)

Initiation of Translation in Prokaryotes: Required Elements

Initiation of translation in prokaryotic cells depends on 3 elements unique to prokaryotes:

• a set of initiation factors (IF 1-3)

• a Shine-Dalgarno sequence on mRNA

• a modified methionine fMet-tRNA^fMet

Two tRNAs for AUG codon (Met)

• the tRNA^fMet is a second tRNA for methionine

• both tRNA^Met (elongation) and tRNA^fMet (initiation) have the same aminoacyl-tRNA synthetase

• tRNA^fMet recruits another enzyme that will modify the amino group of methionine (which then becomes the N-ter amino acid in the synthesized protein)

Initiation of Translation in Prokaryotes: Step 1

• in the first step, a free 30S subunit will bind to IF1 & 3 followed by the binding to mRNA that is guided by the Shine-Dalgarno sequence that positions the first AUG codon in the P site of 30S

• the Shine-Dalgarno sequence is upstream the AUG codon and will hybridize w/ 16S rRNA

• IF3 prevents premature association of the large complex (50S)

• IF1 sits in the A site, so that no undesired tRNA will associate to the complex

• all mRNAs:

  • contain a Shine-Dalgarno sequence in the 5’UTR (upstream of initiation codons)

    • conserved sequences

Initiation of Translation in Prokaryotes: Step 1 FIGURE

Initiation of Translation in Prokaryotes: Step 2

• a GTP bound-IF2 mediates the recruitment of the initiation specific aminoacyl-tRNA in the P site, which is fMet-tRNA ^fMet

Initiation of Translation in Prokaryotes: Step 3

• the large 50S subunit is recruited and following GTP hydrolysis, the three IFs are released

  • 70S complex is formed with mRNA and fMet tRNA

• the complex with the mRNA and fMet-tRNA^fMet in the P site can now proceed to the elongation

Elongation of Translation: Step 1

Recruitment of a charged AA-tRNA in the A site (based on the codon-anticodon hybridization)

• the charged AA-tRNA first binds to Ef-Tu that is associated to GTP (1)

  • EF-Tu mediates entry into A site

  • tRNA don’t freely enter the ribosome, they need elongation factors

• AA-tRNA-Tu (GTP) enters in the A site (2)

• following GTP hydrolysis, Tu(GDP) is released (an exchange factor will then promote association to GTP for a new cycle) (3)

• the AA-tRNA will be ideally repositioned in a stop called accommodation to allow the next step of elongation

Elongation of Translation: Step 1 FIGURE

Elongation of Translation: Step 2

Peptide bond formation occurs in the second step:

• the free amine group of the incoming AA (in A site) attacks the carboxyl group of the previous AA (in P site) (1)

• following the formation of the transient tetrahedral, the first ester bond is cleaved and the preceding AA dissociates from the tRNA in the P-site

• the amine group of the incoming AA forms a peptide bond w/ the preceding AA.

  • The elongated polypeptide in P-site remains associated via its carboxyl end to the incoming tRNA in the A-site.

  • The uncharged (deacetylated) tRNA remains associated to P-site, but shifts toward the E site

Elongation of Translation: Step 2 FIGURE

Elongation of Translation: Step 3 Part 1

• translocation of the ribosome that moves toward the 3’ end of mRNA to position itself for the next round of elongation

• EF-G associated to GTP binds to the A-site as its structure mimics EF-Tu-tRNA (1)

• when EF-G associated in the A site, it will push the ribosome so that the first and second tRNA transition to the E and P sites

Elongation of Translation: Step 3 Part 2

• following the release of EF-G (GDP), the A-site can accept the next charged AA-tRNA (2)

• the uncharged tRNA dissociates from the E site (3)

Elongation of Translation (n-1 cycles)

• the elongation cycle is repeated for each added amino acid

• there are n-1 cycles for a protein of a given length of n AA

• 2 GTPs are required for each cycle

• the elongated polypeptide exits the ribosome via the exit channel in the 50S. The N-ter of the polypeptide is first in the cytosol where it can start to fold

Termination of Translation

• signaled by the presence of one of three stop codons

• presence of a stop codon in the A-site mediates the recruitment of a release factor (RF) that contributes to the hydrolysis of the peptidyl-tRNA bond to release the polypeptide from the ribosome and into the cytosol

• the complex remains associated to mRNA util it can recruit ribosomal recycling factors IF3, EF-G and EFs bound to GTP

• When the ribosome recruits ribosome recycling factor (RRF), along with IF3 and EF-G (bound to GTP), hydrolysis of one final GTP causes the ribosome to split into its 30S and 50S subunits. At the same time, the mRNA, the deacylated (uncharged) tRNA, and the associated factors dissociate

  • two ribosomal subunits, RF, RRF, EF-G (GDP) and the last uncharged tRNA dissociate

• the recycled components can participate to other rounds of translation

• IF3 stays associated to the small subunit

Translation is an RNA-driven Process

The ribosome is a ribozyme as the 23S rRNA (in the 50S subunit) catalyzes peptide bond formation with the help of a 2’OH, while there is no protein within 18ºC A of the active site

• meaning proteins aren’t involved in catalysis

Operon

• an operon is a region of DNA where multiple genes are transcribed together on the same mRNA (often genes that work in the same pathway)

• that mRNA will translate multiple proteins

• each protein has its own start and stop codon sites