translation the relationship between genes and proteins 13 th week gihan e-h gawish, msc, phd ass....
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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