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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’
DNA
5’
rbs
5’ untranslated
AUG
ORF
Open Reading Frame
3’
mRNA
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’end of 16S rRNA
3’ ...UCCUCCA……………………..
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
Termination
when termination codons are reached (UGA, UAA, UAG)
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
anticodon-codon 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