TranslationThe Relationship Between Genes and Proteins

13th Week

Gihan E-H Gawish, MSc, PhDAss. Professor

Molecular Genetics and Clinical Biochemistry

KSU

Table of Contents• Getting from gene to protein: genetic code• Getting from gene to protein: translation

Translation Initiation Translation Elongation Translation Termination References

From gene to protein: genetic code

•Central Dogma Information travels from DNA to RNA to Protein

•Is there a one-to-one correspondence between DNA, RNA and Protein?

–DNA and RNA each have four nucleotides that can form them; so yes, there is a one-to-one correspondence between DNA and RNA.

–Proteins can be composed of a potential 20 amino acids; only four RNA nucleotides: no one-to-one correspondence.

–How then does RNA direct the order and number of amino acids in a protein?

From gene to protein: genetic code• How many bases are required for each amino acid?

• (4 bases)2bases/aa = 16 amino acids—not enough

• (4 bases)3bases/aa = 64 amino acid possibilities

• Minimum of 3 bases/aa required

• What is the nature of the code?

Does it have punctuation? Is it overlapping? Crick, F.H. et al. (1961) Nature 192, 1227–32. (

http://profiles.nlm.nih.gov/SC/B/C/B/J/ ) 3-base, nonoverlapping code that is read from a fixed point.

From gene to protein: genetic code• Nirenberg and Matthaei: in vitro protein translation

Found that adding rRNA prolonged cell-free protein synthesis

Adding artificial RNA synthesized by polynucleotide phosphorylase (no template, UUUUUUUUU) stimulated protein synthesis more

The protein that came out of this reaction was polyphenylalanine (UUU = Phe)

Other artificial RNAs: AAA = Lys; CCC =Pro

From gene to protein: genetic code• Nirenberg:

Triplet binding assay: add triplet RNA, ribosomes, binding factors, GTP, and radiolabeled charged tRNA (figure)

• UUU trinucleotide binds to Phe-tRNA

• UGU trinucleotide binds to CYS-tRNA

By fits and starts the triplet genetic code was worked out.

Each three-letter “word” (codon) specifies an amino acid or directions to stop translation.

The code is redundant or degenerate: more than one way to encode an amino acid

From gene to protein: Translation

• Components required for translation: mRNA Ribosomes tRNA Aminoacyl tRNA synthetases Initiation, elongation and termination factors

Animation

Translation: initiation• Ribosome small subunit binds to mRNA

• Charged tRNA anticodon forms base pairs with the mRNA codon

• Small subunit interacts with initiation factors and special initiator tRNA that is charged with methionine

• mRNA-small subunit-tRNA complex recruits the large subunit

• Eukaryotic and prokaryotic initiation differ slightly

Animation

Translation: initiation• The large subunit of the ribosome contains three binding sites Amino acyl (A site) Peptidyl (P site) Exit (E site)

• At initiation, The tRNAfMet occupies the P site A second, charged tRNA complementary to the next codon binds

the A site.

Translation: elongation• Elongation• Ribosome translocates by three bases after peptide bond formed

• New charged tRNA aligns in the A site• Peptide bond between amino acids in A and P sites is formed

• Ribosome translocates by three more bases• The uncharged tRNA in the A site is moved to the E site.

Translation: elongation

EF-Tu recruits charged tRNA to A site. Requires hydrolysis of GTP

Peptidyl transferase catalyzes peptide bond formation (bond between aa and tRNA in the P site converted to peptide bond between the two amino acids)

Peptide bond formation requires RNA and may be a ribozyme-catalyzed reaction

Translation: termination• Termination• Elongation proceeds until STOP codon reached

UAA, UAG, UGA

• No tRNA normally exists that can form base pairing with a STOP codon; recognized by a release factor

• tRNA charged with last amino acid will remain at P site• Release factors cleave the amino acid from the tRNA• Ribosome subunits dissociate from each other• Review the animation of translation