1. From Gene to Protein How Genes Work

2. from nucleic acid language to amino acid language
Translation
from nucleic acid language to amino acid language

3. Remember the “Central Dogma”
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
Now its time to
focus on translation!
replication

4. Remember:
The Ribosome is the site of protein synthesis – where proteins are MADE
Proteins are POLYMERS
Proteins are made of AMINO ACID MONOMERS
Therefore, if proteins are going to be made, some thing has to bring amino acids to the “protein factory” AND they need to assemble the amino acids into the correct structure to make the needed protein!

5. DETAIL ON TRANSLATION
The completed and processed mRNA molecule goes to the ribosome to be translated.
The image shows the mRNA coming together with the 2 subunits (large and small) that make up the ribosome.
Remember that this occurs in the cytoplasm (rough ER)

6. But how do we know what amino acid goes where???
THE GENETIC CODE
Carried by the mRNA from the DNA
Based on the order of the nucleotides

7. The code Same code is used for ALL life!
strongest support for a common origin for all life
mRNA carries the genetic message from the DNA in the form of codons
Strong evidence for a single origin in evolutionary theory.

8. The Genetic Code 3 nucleotides are required to code for 1 amino acid
The Triplet Code:
The genetic instructions for a polypeptide chain are written in the DNA as a series of non-overlapping, three-nucleotide words.
There are 64 code “words” in the triplet code.
Each triplet is called a CODON.

9. The Genetic Code Each different codon codes for a different amino acid
UGG codes for Trp (tryptophan)
Codons are read by the ribosome in the 5’ to 3’ direction

10. The Genetic Code Special Codons Start Codon Stop Codons Always AUG
Codes for amino acid methionine (met)
Tells ribosome where to begin translating
Thus, “met” is always the first amino acid in any polypeptide
Stop Codons
UAA, UAG and UGA
Tell the ribosome to stop translating
Code for NO amino acids

11. One more player…tRNA
Once the mRNA and ribosome have come together, a third molecule is still required to “interpret” the mRNA message. This is tRNA.
The “t” in tRNA stands for transfer
tRNA transfers amino acids to the ribosome.
There are many tRNA floating about in the cytoplasm.
Each of these tRNA molecule is carrying around one of the 20 different amino acids.
A cell keeps up its supply of amino acids either by
Manufacturing them itself
Taking them up from the surrounding solution

12. Transfer RNA structure
“Clover leaf” structure
anticodon on “clover leaf” end
amino acid attached on 3 end

13. tRNA structure anticodon
This sequence is complementary to one of the different codons on mRNA that code for amino acids and binds to it.

14. Example
If the mRNA codon is UUU, then the tRNA anticodon that will “plug into” this codon is AAA.
Any tRNA molecule with this anticodon (AAA) will always carry the amino acid Phe (phenylalanine) which is what the UUU mRNA codon calls for (see the genetic code chart).

15. Making the polypeptide
As the genetic message is translated, the tRNAs deposit amino acids in the order needed, and the ribosome joins the amino acids into a chain.

16. Loading tRNA w/ correct amino acid
Aminoacyl tRNA synthetase
enzyme which bonds amino acid to tRNA
bond requires energy
ATP  AMP
bond is unstable
so it can release amino acid at ribosome easily
The tRNA-amino acid bond is unstable. This makes it easy for the tRNA to later give up the amino acid to a growing polypeptide chain in a ribosome.
Trp
C=O
Trp
Trp
C=O OH
H2O OH O
C=O O
activating
enzyme
tRNATrp A C C U G G
mRNA
anticodon
tryptophan attached to tRNATrp
tRNATrp binds to UGG condon of mRNA

17. How do amino acids get hooked to the correct tRNAs?
There is an enzyme called aminoacyl-tRNA synthetase – actually there are 20 versions of this enzyme – one for each of the 20 different amino acids
The active sites of each of these enzymes fits only a specific combination of amino acid and tRNA
Once bound, the synthetase catalyzes the covalent attachment of an amino acid to its appropriate tRNA
This process is driven by ATP.

18. Ribosomes Facilitate coupling of tRNA anticodon to mRNA codon
organelle or enzyme?
Structure
ribosomal RNA (rRNA) & proteins
2 subunits
large
small E P A

19. Ribosomes A site (aminoacyl-tRNA site) – ACTIVE site
holds tRNA carrying next amino acid to be added to chain
P site (peptidyl-tRNA site) – PASSIVE site
holds tRNA carrying growing polypeptide chain
E site (exit site)
empty tRNA leaves ribosome from exit site
Met U A C 5' U G A 3' E P A

20. rRNA or Protein – which one makes the ribosome work?
Ribosomes are made of both
rRNA and
protein
rRNA is most responsible for ribosomal structure and function
Ribosome is one big ribozyme
RNA acting as catalyst

21. Building a polypeptide1 2 3
Building a polypeptide
Initiation
brings together mRNA, ribosome subunits, initiator tRNA
Elongation
adding amino acids based on codon sequence
Termination
end 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'

22. Translation - Initiation
Components brought together
mRNA
Two ribosomal subunits
tRNA
Bearing the first amino acid of the polypeptide that will be made
Remember this is always “met”
met

23. Translation - Initiation
mRNA attaches to
small ribosomal subunit and…
tRNA carrying “met” amino acid
Small subunit then moves downstream along the mRNA until it encounters the start codon (AUG)
This establishes the reading frame.
The anticodon of the initiator tRNA (w/ “met”) then hydrogen bonds with the start codon on mRNA

24. Translation - Initiation
After mRNA, tRNA and small subunit unite…
Large ribosomal subunit attaches
This completes the translation initiation complex
Initiation factors
Proteins
Bring all components together
Energy used
GTP
At this point the initiator tRNA is sitting on the P site of the ribosome.
The A site is vacant and waits for the next tRNA to arrive

26. Translocation - Elongation
3 Parts
Codon Recognition
Peptide Bond Formation
Translocation

27. Translation - Elongation
Codon recognition
Anitcodon of incoming tRNA base pairs with the complementary mRNA sequence which is at the A site of the ribosome

28. Codon recognition

29. Translation - Elongation
Peptide bond formation
Peptide bond is formed between the new amino acid at the A site and the old amino acid that has been waiting at the P site
This step removes the amino acid from the tRNA at the P site and adds it to the amino acid at the A site
Thus the polypeptide chain begins to grow

30. Peptide bond
formation

31. Translation - Elongation
Translocation
tRNA moves from the A site to the P site
tRNA at the P site moves to the E site where it is released
mRNA moves along to bring the next codon to the A site.

32. Translocation –
Everything shifts
over

33. Translation - Termination
Elongation continues until a stop codon is reached
UAA, UAG, UGA
Release factor
Protein
Binds to stop codon at A-site
Causes addition of water instead of amino acid to polypeptide
Releases the polypeptide from the tRNA at the P-site
Remainder of the whole assembly comes apart

34. Polyribosomes
Multiple ribosomes translating the same piece of mRNA

35. Modifications of Polypeptides to make FUNCTIONAL Proteins
During synthesis, polypeptides begin to fold spontaneously
Creates specific shape (conformation)
Structure
Gene determines primary structure
Primary structure determines conformation
Chaperone proteins
May help the protein to fold correctly
Post-translational modifications
Amino acids may be modified, removed
Polypeptide may be cleaved
2 or more polypeptides may come together (quaternary structure)

37. start of a secretory pathway
Destinations:
secretion
nucleus
mitochondria
chloroplasts
cell membrane
cytoplasm
etc…
Protein targeting
Signal peptide
address label
start of a secretory pathway

38. Multiple Roles of RNA in Cells
mRNA - carries information specifying amino acid sequences of proteins from DNA to ribosomes.
tRNA - translates mRNA codons into amino acids
rRNA - plays catalytic (ribozyme) AND structural roles in ribosomes.
Primary transcript - precursor to mRNA (or rRNA or tRNA), before being processed by splicing, etc.
Small nuclear RNA (snRNA)- plays structural and catalytic roles in spliceosomes.
SRP RNA - component of the SRP that recognizes the signal peptides of polypeptides targeted to ER.
Small nucleolar RNA- aids in process pre-rRNA transcripts for making ribosome subunits in the nucleolus.
Small interferring RNA and microRNA - involved in determining which genes get expressed in a cell.

39. How Come RNA Can Do So Many Different Things?
Because of 3 properties:
RNA can hydrogen bond to other nucleic acids
RNA can assume a specific 3-dimensional shape
These can form hydrogen bonds between bases in different parts of the RNA’s own nucleotide chain
Results in complex folding
RNA has functional groups that allow it to act as a catalyst (ribozymes)
Bottom Line: RNA is much more versatile than DNA

40. How does mRNA code for proteins?
TACGCACATTTACGTACGCGG
DNA 4
ATCG
AUGCGUGUAAAUGCAUGCGCC
mRNA 4
AUCG ?
Met Arg Val Asn Ala Cys Ala
protein 20
How can you code for 20 amino acids with only 4 nucleotide bases (A,U,G,C)?

41. mRNA codes for proteins in triplets
TACGCACATTTACGTACGCGG
DNA
codon
AUGCGUGUAAAUGCAUGCGCC
mRNA
AUGCGUGUAAAUGCAUGCGCC
mRNA ?
Met Arg Val Asn Ala Cys Ala
protein

42. How are the codons matched to amino acids?3 5
DNA
TACGCACATTTACGTACGCGG 5 3
mRNA
AUGCGUGUAAAUGCAUGCGCC
codon 3 5
UAC
Met
GCA
Arg
tRNA
CAU
Val
anti-codon
amino acid

43. DNA mRNA protein trait From gene to protein nucleus cytoplasmaa
From gene to protein
nucleus
cytoplasm
transcription
translation
DNA
mRNA
protein
ribosome
trait

44. Can you tell the story? RNA polymerase DNA amino acids tRNA pre-mRNA
exon
intron
tRNA
pre-mRNA
5' GTP cap
mature mRNA
aminoacyl tRNA synthetase
poly-A tail 3'
large ribosomal subunit
polypeptide 5'
tRNA
small ribosomal subunit E P A
ribosome