Lecture 3
Gene Structure, Transcription, & Translation
Reading:
Chapter 4: 108-115; 118-131
Molecular Biology syllabus web site
Typical Gene Structure
Promoter Coding Region
+1
transcription
Prokaryotes
COORDINATED GENE EXPRESSION: clustered genes (operon) controlled by one promoter and transcribed as polycistronic mRNA and encode multiple gene products
Eukaryotes
Interrupted genes (exons/introns)
Monocistronic mRNAs
Post-transcriptional modifications (nuclear encoded genes): 5 CAP
polyA tail
splicing
Post-transcription addition of 5 CAP to nuclear encoded eukaryotic mRNA
Transcript Structure
3 5 5
rbs
AUG
DNA
ORF
Open Reading Frame
mRNA
5 untranslated
3 untranslated
protein
Transcription: RNA Synthesis
1. 2. 3. 4.
5.
Requirements Enzyme: RNA Polymerase DNA Template (3 to 5 strand) No primer required Nucleoside triphosphates: ATP, GTP, CTP, UTP Synthesis is 5 to 3
Transcription: RNA Synthesis
Translation: Protein Synthesis
Codons specify amino acids; positioning on ribosome sets READING FRAME
The roles of RNA in protein synthesis
Copyright (c) by W. H. Freeman and Company
The three roles of RNA in protein synthesis
Three types of RNA molecules perform different but complementary roles in protein synthesis (translation) Messenger RNA (mRNA) carries information copied from DNA in the form of a series of three base words termed codons Transfer RNA (tRNA) deciphers the code and delivers the specified amino acid Ribosomal RNA (rRNA) associates with a set of proteins to form ribosomes, structures that function as proteinsynthesizing machines
Copyright (c) by W. H. Freeman and Company
The folded structure of tRNA specifies its decoding function
Figure 4-26
Copyright (c) by W. H. Freeman and Company
Aminoacyl-tRNA synthetases activate amino acids by linking them to tRNAs
Each tRNA molecule is recognized by a specific aminoacyltRNA synthetase
Fidelity of protein synthesis determined by:
Correct aminoacylation of tRNA Codon-anticodon pairing
Aminoacyl tRNA synthetases
-at least one for every amino acid -for different codons have different synthetases -error correction lies in specificity of synthetase and tRNA. No mechanism exists for error correction once tRNA is mischarged and separated from synthetase
Double sieve mechanism for error correction
Synthetases have 2 sites: active site, hydrolytic site. Amino acids larger than the correct amino acid are never activated because they are too large to fit into the active site. Smaller amino acids (than the correct one) fit into the hydrolytic site (which excludes the correct amino acid) and are hydrolyzed.
Nonstandard base pairing often occurs between codons and anticodons
Ribosomes: the macromolecular site for protein synthesis
Translation
Initiation Elongation Termination
Initiation
mRNA binds to ribosome Selection of initiation codon Binding of charged initiator tRNA (first amino acid)
Initiation
Formation of 30S preinitiation complex 30 S subunit (contains 16S rRNA), mRNA, charged tRNA f-met, initiation factors, GTP + 50S subunit (GTP hydrolysis) Resulting in formation of the 70S initiation complex fmet-tRNA is fixed into the P site reading frame is now determined.
Initiation
Initiation of eukaryotic protein synthesis generally occurs at the 5 end of mRNA but may occasionally occur at internal sites
Initiation of prokaryotic protein synthesis generally occurs at the Shine Delgarno site
The untranslated leader or 5 end of prokaryotic mRNAs contain a ribosome binding site (rbs) or Shine Delgarno site located upstream of the AUG and complementary to the 3 end of the 16S rRNA.
mRNA:
5 .AGGAGGU..AUG 3 ...UCCUCCA..
3end of 16S rRNA
IIIIIII
Elongation
Peptide bond formation Movement of mRNA/ ribosome (translocation) so each codon may be read
Elongation
Requirements: Elongation factors and GTP hydrolysis
Occupation of A site by next tRNA
Peptide bond formed by peptidyl transferase enzyme
Uncharged tRNA-fmet in P site and dipeptidyl tRNA in A site Translocation:
deacylated tRNA fmet leaves P site
peptidyl tRNA moves from A to P site mRNA moves 3 bases to position next codon at A site
Elongation
when termination codons are reached (UGA, UAA, UAG)
Termination
Completed protein is dissociated from machinery Ribosome released
Termination
when termination codons are reached (UGA, UAA, UAG)
Peptidyl tRNA moves from A to P site Release factors (RF) recognize specific stop codons
RF forms activated complex with GTP
Activated complex binds to termination codon and alters specificity of peptidyl transferase
In presence of RF, peptidyl transferase catalyzes reaction of bound peptidyl moiety with water instead of with free aminoacyl tRNA Release of polypeptide
Dissociation of 70S ribosome into 50S and 30S subunits.
Summary of Protein Synthesis
1. Binding of mRNA to ribosome
2. Charged, amino-acylated initiator tRNA binds to P site of ribosome and is based paired through tRNA anticodon to codon on mRNA
3. A second amino-acylated tRNA fills A site and anticodon Hbonds with second codon on mRNA 4. Amino acids in P and A site are joined by a peptide bond. tRNA in P site is released. tRNA (with 2 amino acids joined) in A site moves to P site A new amino-acylated tRNA moves into A site by anticodoncodon pairing 5. Step (4) is repeated until codon in A site is a stop codon; peptide is released.
Post-translational Modifications
(Bacteria) removal of formyl groups (fmet) removal of first few amino acids (aminopeptidase) glycosylation (affects targeting, activity) phosphorylation (by kinases) S-S bond formation Polypeptide cleavage -removal of transit peptide upon organelle import -removal of signal sequence (ER secretion) -activation of enzymes
