From RNA to protein

Kanokporn Boonsirichai

The coding problem How is the information in a linear sequence of

nucleotides in mRNAs translated into the linear sequence of amino acids in proteins?

DNA Deoxynucleotides

RNA Ribonucleotides

Protein Amino acids

4 kinds

4 kinds

20 kinds

Molecules Monomers Monomer variations

The Genetic Code

mRNA sequence is decoded in a set of three nucleotides

For a sequence, there exists three possible reading frames.

tRNA

The code is translated as a triplet of three nucleotides into the corresponding amino acid via a transfer RNA.

Codon

Anticodon

Structure of a tRNA

Redundancy of the code

More than one tRNAs for some amino acids

Some tRNAs can base pair with more than one codon

tRNA Biosynthesis Synthesized by RNA polymerase III

Synthesized as a larger precursor

- trimmed

- spliced

- base modifications

tRNA Splicing

Some precursor-tRNAs are with introns

tRNAs introns are cut and pasted together by endonuclease and tRNA ligaseCut within the tRNA

Paste the tRNA pieces together

Coupling of amino acid to tRNAs

Achieved by aminoacyl-tRNA synthetases

Aminoacyl-t-RNA synthethase

How is accuracy achieved?

The correct amino acid has the highest affinity for the active site.

Hydrolytic editing

Nucleotide binding pockets/ acceptor stem recognition

1 mistake in 40,000 tRNA couplings

tRNAGln

Synthesis of Proteins

Formation of a peptide bond between the carboxyl group at the end of a growing polypeptide chain and a free amino group on an incoming amino acid

The synthesis is stepwise from the N-terminal end to the C-terminal end.

The growing carboxyl end of the polypeptide chain remains activated by its covalent attachment to a tRNA molecule (a peptidyl-tRNA molecule).

Peptidyltransferasereaction

Decoding mRNAs 1 mistakes in 10,000 amino acid polymerized

2 amino acids are polymerized per second in eukaryotes

20 amino acids are polymerized per second in prokaryotes

So, where is it done?

What do you on this micrograph?

Ribosome

Ribosome subunits and their rRNAs are assembled in the nucleolus and exported to the cytoplasm where translation occurs.

Translation Elongation

A ribosome contains four binding sites for RNA molecules: 3 for tRNAs and 1 for mRNA.

tRNA anticodon interacts with the codons on the mRNA in the small subunit of the ribosome.

The large subunit catalyzes the peptidyl transferase reaction.

A-site: Aminoacyl-tRNA

P-site: Peptidyl-tRNA

E-site: Exit site

Translation Elongation

Elongation factors and translation

accuracy

Ribosomes Contain 2/3 RNAs and 1/3 proteins

rRNAs are folded into a highly compact three dimensional structure responsible for the overall shape of the ribosome

23S rRNA forms the catalytic site

Translation Initiation Initiator tRNAs always carry the amino acid

methionine.

The small ribosomal subunit scans the mRNA in the 5’ to 3’ direction .

Nucleotides immediately surrounding the start site in eucaryotic mRNAs influence the efficiency of AUG recognition.

Bacterial mRNAs are polycistronic

Specific sequence for ribosome binding: Shine-Dalgarno sequence

Located 8 bp upstream of the start codon.

Translation Termination

Stop codons (UAA, UGA, UAG) signal the ribosome to stop translation

Release factors bind to the ribosome at the A-site causing H2O to be added to the growing peptide chain, releasing it from attachment with the tRNA.

eRF1 and tRNA structure comparison

Polysomes: multiple ribosomes on a single mRNA Protein synthesis takes 20 seconds to several

minutes

Ribosomes are spaced ~80 nt apart on an mRNA

Translation Accuracy

1 mistake per 10,000 amino acids joined

20 amino acids are incorporated per second in bacteria.

Translation is a compromise between accuracy and speed.

If too slow, cannot produce enough proteins to survive.

Price of Protein Synthesis

Four high-energy phosphate bonds must be split per peptide bond.

Two to charge the tRNA with an amino acid

Two to drive the synthesis itself on the ribosome

Extra energy is consumed for incorrectly charged tRNA and for entrance of an incorrect tRNA into the ribosome.

Quality control features Both the 5’ cap and the poly-A tail must be

recognized before an mRNA enters the ribosome.

Why?