The ONE (Translation I & II)

Genetic code

The peptide produced was observed and the code was determined.

What was the genetic code designed for?

Minimize impact of single nucleotide mutations

(Mutations at 3rd position (wobble) can be silent or if not will conserve size polarity if the encoded amino acid.

Frameshift

Code is shifted by 1 or 2 bases.

What does a frameshift allow for?

Allow for a modified reading frame to produce a unqie peptide sequence

Translation @ Ribosome

Energy drive, template based polymer synthesis, executed through sophisticated molecular recognition events

Speed of Translation @ ribosome

7-20 amino acids per second

Accuracy of translation @ Ribosome

One mistake per 1000-10000 aminoacids incorporated

Ribosome

Mega strcuture comprised of assembled folded RNA (ribosomal=rRNA) and small proteins

Participants in Translation include

- tRNA

-20 amino acids

-ATP

-Aminoacyl tRNA synthetases

-mRNA= code

- Numerous Protein facotrs

- GTP

what are the 3 tRNA binding sites of the Ribosome?

- Exit site "E"

- Peptidyl Site "P"

- Amino Acyl Site "A"

Exit Site "E"

The used tRNA (aa removed) departs from this site which is located Primarily on the 50S unit

Peptidyl Site (P)

At initiation the tRNA (fMET) binds here At elongation the tRNA (peptide) binds here

AminoAcyl Site "A"

At elongation where the entering tRNA(aa) binds

The tRNA has two functional sites

- the site at which the amino acid is attached via an ester linkage

- site at which it binds to the codon à this site is called the anticodon

Aside from Translation 3 phases (initation, elongation, and termination) what are the 2 other steps?

- Charging of tRNA

- Posttranslational modification

Step 1: Activation of the tRNA for translation

- Amino acid must be tethered to the tRNA, mediated by tRNA synthetase (1 ATP consumed)

Discrimination in charging

Accurate selection of the cognate tRNA from the cellular pool by an synthase is know to involve a dual discrimination mechanism based upon velocity of the aminoacylation reaction and substrate binding affinity

tRNA cloverleaf

three regions that are important for interactions with and binding to the appropriate synthase. The acceptor stem, the D loop and the anticodon loop

IF-1

Prevents premature binding of tRNAs to A site

IF-2

Facilitates binding of fMet-tRNA to 305 ribosomal subunit

IF-3

Binds to 30s subunit; prevents premature association of 505 subunit; enhances specificity of P site for fMet-tRNA

Step 2: Initiation Complex

Initiation factors guide the ribosome in the selection of mRNA and translational reading frame. IF3and IF2 are the first factors to arrive, forming an unstable 30S-IF2_IF3complex. Subsequently, IF1joins and locks the factors in a kinetically stable 30S PIC to which fMET-tRNAfmet is recruited. Bindingof mRNA is independent of initiation factors and can take place at any time during 30S PIC assembly

what does IF1 do in the initiation complex?

Binds to the A site blocking it so the first aa-tRNA will bind to the p Site

what does IF3 do in the initiation complex?

Binding prevents binding 50s binding to 30s before mRNA has a chnace to bind.

Step 2: Initiation complex where to start?

In bacteria a purine stretch of sequence just upstream of the initiation codon base pairs with with a complementary pyrimidine rich stretch of sequence on the 30S unit

when fMet-tRNA cannot bind to the A site becuase it is blocked by IF1?

It binds to the P-site by "special interactions" and to the AUG codon (anticodon)

To make Initiation complex ACTIVATE

50s binds, GTP on IF2 is hydrolyzed, all 3 IFs dissociate. The "A" site is open to bind a charged tRNA to imitate elongation

Step 3: Elongation

Elongation factors are important for this step are EF-Tu and EF-G

What must occur before Elongation starts?

Ribosome structure is bound with the initial tRNA and mRNA

What happens after completion of aminoacylation?

Most charged tRNA species are sequestered by EF-Tu and subsequently proceed into protein synthesis ar the ribosome.

Step 3: Elongation: Loading the aminoacyl-tRNA in A site

- EF-Tu-tRNA complex binds to the A site of the ribosome

- Hydrolysis of GTP->GDP initiates dissociation of the EF-Tu tRNA complex, allowing the free tRNA to slide into place with the peptidyl tRNA in the Psite.

- Ef-Tu is repurposed with regeneration of the GTP to bind another charged tRNA

Step 3: Elongation: production of peptide bond

Peptide bond formation is catalyzed by the peptidyl transferase center

Step3: Translocation

- GTP hydrolysis precedes and accelerates translocation.

- The EF-G bound GTP binds to the Asite and catalyzes the translocation

What must occur for translocation to move forawrd along the mRNA?

Unclamping/unlocking of the ribosome.

Step 4: Termination

- Termination (stop) codon (UAA, UAG,UGA) follows final reading codon and signals the end of the translation

- The release factor (RF1 or RF2) binds tothe stop codon at the A site. The peptide of the peptidyl-tRNA is transferred towater then leaves the ribosome as the new protein. RF3 has unknown function.

-The 70S dissociates to 50S and 30S with the help of the EF-G, RRF, and IF3

-The mRNA (t 1/2 1 min) will be degraded by enzymes called nucleases

Step 4: Termination recognition

The hydrogen bonds serve as the recognition element -it is critical that RF1/RF2 bind only with their respective stop codon recognized

how does RF1 stimulate the transfer of the peptide unit from the peptidyl-tRNAto a water molecule?

binds to the reaction center and the Gly229 backbone amide NH stabilizes the transition state that is formed upon attack of water at the ester group.

what is the "Mark of death"?

Ubiquitin

What does Ubiquitin do?

Signifies the protein for degradation by proteosome.

What is the "mark of death" labelled as when it is repeated?

conjugation