1. Gene Structure, Transcription, & Translation
Lecture 3
Gene Structure, Transcription, & Translation
Reading: Chapter 4: ;
Molecular Biology syllabus web site

3. Typical Gene Structure
Promoter
Coding Region +1
transcription

4. Prokaryotes
COORDINATED GENE EXPRESSION: clustered genes (operon) controlled by one promoter and transcribed as polycistronic mRNA and encode multiple gene products

5. Eukaryotes Interrupted genes (exons/introns) Monocistronic mRNAs
Post-transcriptional modifications (nuclear encoded genes):
5’ CAP
polyA tail
splicing

6. Post-transcription addition of 5’ CAP to nuclear encoded eukaryotic mRNA

7. Transcript Structure 3’ 5’ 5’ 3’ ORF rbs DNA mRNA AUG
3’ ’
5’ ’
DNA
mRNA
rbs
AUG
ORF
Open Reading Frame
3’ untranslated
5’ untranslated
protein

11. Transcription: RNA Synthesis
Requirements
Enzyme: RNA Polymerase
DNA Template (3’ to 5’ strand)
No primer required
Nucleoside triphosphates: ATP, GTP, CTP, UTP
Synthesis is 5’ to 3’

12. Transcription: RNA Synthesis

13. Codons specify amino acids; positioning on ribosome sets READING FRAME
Translation: Protein Synthesis
Codons specify amino acids; positioning on ribosome sets READING FRAME

16. The roles of RNA in protein synthesis
Copyright (c) by W. H. Freeman and Company

17. 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 protein-synthesizing machines
Copyright (c) by W. H. Freeman and Company

18. The folded structure of tRNA specifies its decoding function
Figure 4-26
Copyright (c) by W. H. Freeman and Company

19. Aminoacyl-tRNA synthetases activate amino acids by linking them to tRNAs
Each tRNA molecule is recognized by a specific aminoacyl-tRNA synthetase

20. Fidelity of protein synthesis determined by:
Correct aminoacylation of tRNA
Codon-anticodon pairing

21. Double sieve mechanism for error correction
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.

22. Nonstandard base pairing often occurs between codons and anticodons

24. Ribosomes: the macromolecular site for protein synthesis

25. Translation
Initiation
Elongation
Termination

26. Initiation mRNA binds to ribosome Selection of initiation codon
Binding of charged initiator tRNA (first amino acid)

27. 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.

28. Initiation

31. Initiation of eukaryotic protein synthesis generally occurs at the 5’ end of mRNA but may occasionally occur at internal sites

32. 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……………………..
I I I I I I I

35. Elongation Peptide bond formation
Movement of mRNA/ ribosome (translocation) so each codon may be “read”

36. 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

37. Elongation

42. when termination codons are reached (UGA, UAA, UAG)
Completed protein is dissociated from machinery
Ribosome released

43. 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.

44. 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 H-bonds 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.

45. 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