1. Protein synthesis

2. Big Picture Make a copy of DNA (in nucleus)
Send copy out of nucleus into cytoplasm
Read copy on ribosome
Make a protein
“Central Dogma”

3. Organelles and cell machinery involved in protein synthesis
Nucleus & Nucleolus
Ribosomes
Endoplasmic Reticulum
Golgi Apparatus
Vesicles

4. The Central Dogma (aka “the big picture”)
Flow of genetic information in a cell
How do we move information from DNA to proteins?
Transcription
Translation
DNA
RNA
PROTEIN
TRAIT
To get from the chemical language of DNA to the chemical language of proteins requires 2 major stages:
transcription and translation
Replication

6. Here we go…. Stage 1: Transcription
Goal
Make a copy of DNA in the form of RNA
Location
NUCLEUS

7. Transcription Anatomy: DNA to mRNA
Transcription Unit (GENE): segment of DNA to be transcribed
transcribed DNA strand = Template Strand
Only read1 strand (template strand)…make complimentary mRNA strand
transcription bubble
enzyme
RNA Polymerase
coding strand 5 3 A G C A T C G T A G A A
DNA G T C A T T C T C 3 A T A C G T C G 3 T A A T 5 G G C A U C G U T C
unwinding G T A G C A
rewinding
mRNA 5
RNA polymerase
template strand

8. Basic structure of a protein encoding gene:
DNA
Promoter: identified by RNA poly…attach
Gene: actual DNA turned into mRNA
Terminator: RNA poly detaches

9. Transcription: Initiation
Eukaryotes
Transcription Factors
Protein helpers
Help RNA poly attach to promoter
Looks for TATA BOX
TATA Box
Upstream end of promoter region

10. Transcription: Elongation
RNA Polymerase moves along DNA, opening bases at a time
Adds new RNA Nt to the growing 3’ end
RNA strand peels away & DNA helix reforms
reads DNA 35

11. Transcription: Elongation
Animations:

12. Question
What would be the complementary RNA strand for the following DNA sequence?
DNA 5’-GCGTATG-3’

13. Transcription: termination
Eukaryotes
RNA Polymerase transcribes a “signal” (AAUAAA) in terminator region of gene
END

14. Almost done… Stage 2: Translation
Goal
Read mRNA and turn into a PROTEIN
mRNA
Goal
RIBOSOME (cyto or RER)
Ribosome

15. Ribosomes Structure ribosomal RNA (rRNA) & proteins 2 subunits large
small

16. Ribosomes A site (accepting site) P site (protein syn site)
holds tRNA carrying next amino acid to be added to chain
P site (protein syn site)
holds tRNA carrying growing polypeptide chain
E site (exit site)
empty tRNA leaves ribosome from exit site

17. Codon Chart mRNA is read in sets of 3 bases…CODON
CODON codes for Amino Acid
Start codon
AUG
methionine
Stop codons
UGA, UAA, UAG

18. Primary structure of a protein
Messenger RNA (mRNA) A U G C
mRNA
start
codon
codon 2
codon 3
codon 4
codon 5
codon 6
codon 7
codon 1
methionine
glycine
serine
isoleucine
alanine
stop
codon
protein
Primary structure of a protein
aa1
aa2
aa3
aa4
aa5
aa6
peptide bonds

19. Yikes…here come the details!!

20. Building a polypeptide1 2 3
Initiation
brings together mRNA, ribosome subunits, initiator tRNA
Elongation
adding amino acids based on codon sequence
Termination
stop codon
Leu
Val
release
factor
Ser
Met
Met
Met
Met
Leu
Leu
Leu
Ala
Trp
tRNA C A G U A C U A C G A C A C G A C A 5' U 5' U A C G A C 5' A A A U G C U G U A U G C U G A U A U G C U G A A U 5' A A U
mRNA A U G C U G 3' 3' 3' 3' A C C U G G U A A E P A 3'

21. Translation: Initiation
Small ribosomal subunit binds to mRNA
Start codon AUG= methionine AA
Large ribosomal subunit binds
Closes down on small

22. Translation: Elongation
tRNA brings in A.A and it sits in A site
Enzyme in the large subunit of the ribosome catalyzes a peptide bond between new AA in A site and AA in P site
Ribsome moves the A site tRNA to the P site
Empty tRNA at P site ejected from E site
Moves codon to codon 5’ to 3’ on mRNA and builds polypeptide from N-terminus to C-terminus

23. Translation: Termination
Stop codon reaches A site
Release factors bind to A site
Polypeptide released

24. Translation Animations

25. DNA 3’-TCGTACGGGATACCCAAATATCGAACTCTC-5’
Question
What would be the complementary mRNA strand and amino acid sequence for the following DNA sequence?
DNA 3’-TCGTACGGGATACCCAAATATCGAACTCTC-5’
mRNA: 5’AGCAUG-CCC-UAU-GGG-UUU-AUA-GCU-UGAGAG 3’
tRNA anticodons: UAC-GGG-AUA-CCC-AAA-UAU-CGA-ACU
Polypeptide: met-pro-tyr-gly-phe-ile-ala
mRNA: 5’AGCAUG-CCC-UAU-GGG-UUU-AUA-GCU-UGAGAG 3’
tRNA anticodons: UAC-GGG-AUA-CCC-AAA-UAU-CGA-ACU
Polypeptide: met-pro-tyr-gly-phe-ile-ala

26. Transfer RNA (tRNA) structure
“Clover leaf” structure
Anticodon on “clover leaf” end
Complementary to mRNA codon
Amino Acid attached on 3 end

27. Practice all of Protein Synthesis

28. Compare the 3 types of RNA

29. Prokaryote vs Eukaryote

30. Ribosomes: prokaryotes vs. eukaryotes

31. Prokaryote
Transcription initiation: RNA Polymerase recognizes promoter and binds…no transcription factors needed

32. Transcription in Eukaryotes
3 RNA polymerase enzymes
RNA polymerase 1
only transcribes rRNA genes
makes ribosomes
RNA polymerase 2
transcribes genes into mRNA
RNA polymerase 3
only transcribes tRNA genes
each has a specific promoter sequence it recognizes

33. Translation in Prokaryotes
Transcription & translation are simultaneous in bacteria
DNA is in cytoplasm
no mRNA editing
ribosomes read mRNA as it is being transcribed

34. What happens to the protein when the DNA is wrong??

35. Types of Mutations 2 categories: Base – pair substitution
Base – pair insertions or deletions

36. Base-Pair Substitution
Replacement of one nucleotide
Silent mutations: do not present a change in protein
(multiple codons for one amino acid)
Missense mutations: still code for an amino acid; but the wrong amino acid.
Nonsense mutation: codes for a STOP CODON – translation ends prematurely

37. Insertions & Deletions
Additions or losses of nucleotide pairs in a gene
Have deleterious effects b/c alter the “reading frame” of the genetic message = frameshift mutation
Occurs whenever the insertion or deletion is NOT a multiple of three

38. Regulating protein synthesis

39. Regulating at DNA level

40. Histone Acetylation
Histone acetylation turns genes = ON
attachment of acetyl groups (–COCH3) to positively charged lysines (neutralize AA)
when histones are acetylated they change shape & grip DNA less tightly = unwinding DNA
transcription proteins have easier access to genes

41. DNA packing & methylation
Chromatin modifications affect the availability of genes for transcription
DNA methylation turns genes = off
attachment of methyl groups (–CH3) to DNA bases (cytosine) after DNA is synthesized
nearly permanent suppression of genes
ex. the inactivated mammalian X chromosome

42. Regulating at transcription level

43. Post-transcriptional processing
Primary transcript (pre-mRNA)
eukaryotic mRNA needs work after transcription
mRNA processing (making mature mRNA)
mRNA splicing = edit out introns (non-coding regions)
protect mRNA from enzymes in cytoplasm
add 5’ G cap
add 3’ Poly A tail
3' poly-A tail 3' A A A A A
5' cap
mRNA
A’s P P P 5' G
intron = noncoding (inbetween) sequence
eukaryotic RNA is about 10% of eukaryotic gene.
~10,000 bases
eukaryotic DNA
exon = coding (expressed) sequence
pre-mRNA
primary mRNA
transcript
~1,000 bases
mature mRNA
transcript
spliced mRNA

44. Splicing enzymes snRNPs Splicesome several snRNPs
small nuclear RNA
proteins
Splicesome
several snRNPs
recognize splice site sequence
cut & paste
snRNPs
exon
intron
snRNA 5' 3'
spliceosome
exon
excised
intron 5' 3'
lariat
mature mRNA
Prokaryotes do not have introns
Not all genes have introns: histones do not
Dystrophin gene (99% introns)
No, not smurfs!
“sNurps”

45. mRNA Modification Animations

46. Regulating at translation level