1. DNA to Protein
Genomic Expression

2. DNA to Protein
DNA acts as a “manager” in the process of making proteins
DNA is the template or starting sequence that is copied into RNA that is then used to make the protein
Nobel Prize website:

3. Central Dogma: One Gene – One Protein
This is the same for bacteria to humans
DNA is the genetic instruction or gene
DNA  RNA is called Transcription
RNA chain is called a transcript
RNA  Protein is called Translation

4. Genes A string of codons codes for several amino acids to form a gene
A gene can be as short as 50 nucleotides and as long as 250 million.
Humans have over 3 billion nucleotides or 1 billion codons
Each gene codes for a certain trait.

5. Expression of Genes
Some genes are transcribed in large quantities because we need large amount of this protein
Some genes are transcribed in small quantities because we need only a small amount of this protein

6. Products of Transcription
Ribosomal RNA (rRNA) – Carrier molecule on ribosomes – holds the tRNA and mRNA in place
Transfer RNA (tRNA) - The molecule that physically couples nucleic acid codons with specific amino acids
Messenger RNA (mRNA) - The messenger that carries information from genes on DNA to the protein manufacturing ribosomes

7. Transcription DNA is copied to RNA T is changed to a U
So then A bonds with a U (Uracil)
Proceeds in the 5’-3’ position
mRNA – leaves nucleus as a copy and codes for an amino acid (translation)

8. Translation occurs within the cytoplasm of cell tRNA – transfer RNA
decodes information from mRNA to produce amino acids
3 codons translate to an amino acid
Translation animation

9. Amino Acid
A chain of nucleotides makes a codon (3 letter word such as ATT, GCA
Each codon makes an amino acid (20 essential Amino Acids)
“Stop” codons means translation stops and a gene is complete

10. Protein Synthesis occurs on Ribosomes

11. Translation The genetic message encoded in RNA is used to create a polypeptide with a specific amino acid sequence
This is a molecule of messenger RNA. It was made in the nucleus by transcription from a DNA molecule.
Codon= series of three nucleotides
A U G G G C U U A A A G C A G U G C A C G U U
mRNA molecule

12. Messenger RNA (mRNA) Primary structure of a protein
Each codon translates into one of twenty amino acids or is a stop or start signal 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

13. The Genetic Code UUU UUC UUA UUG CUU CUC CUA CUG AUU AUC AUA AUG GUU
GUC
GUA
GUG
UCU
UCC
UCA
UCG
CCU
CCC
CCA
CCG
ACU
ACC
ACA
ACG
GCU
GCC
GCA
GCG
UAU
UAC
UAA
UAG
CAU
CAC
CAA
CAG
AAU
AAC
AAA
AAG
GAU
GAC
GAA
GAG
UGU
UGC
UGA
UGG
CGU
CGC
CGA
CGG
AGU
AGC
AGA
AGG
GGU
GGC
GGA
GGG
Phenylalanine
Leucine
Valine
Serine
Proline
Threonine
Alanine
Tyrosine
Stop
Histidine
Glutamine
Asparagine
Lysine
Glutamic
Acid
Cysteine
Arginine
Serine
Glycine
Stop
Tryptophan
Isoleucine
Methionine

14. Note that 3rd Base Position is Variable

15. Transcription
Copy the gene of interest into RNA which is made up of nucleotides linked by phosphodiester bonds – like DNA
RNA differs from DNA
Ribose is the sugar rather than deoxyribose – ribonucleotides
U instead of T; A, G and C the same
Single stranded
Can fold into a variety of shapes that allows RNA to have structural and catalytic functions

16. RNA Differences

17. RNA Differences

18. Transcription Similarities to DNA replication Differences
Open and unwind a portion of the DNA
1 strand of the DNA acts as a template
Complementary base-pairing with DNA
Differences
RNA strand does not stay paired with DNA
DNA re-coils and RNA is single stranded
RNA is shorter than DNA
RNA is several 1000 bp or shorter whereas DNA is 250 million bp long

19. Template to Transcripts
The RNA transcript is identical to the NON-template strand with the exception of the T’s becoming U’s

20. RNA Polymerase
Catalyzes the formation of the phosphodiester bonds between the nucleotides (sugar to phosphate)
Uncoils the DNA, adds the nucleotide one at a time in the 5’ to 3’ fashion
Uses the energy trapped in the nucleotides themselves to form the new bonds

21. RNA Elongation Reads template 3’ to 5’
Adds nucleotides 5’ to 3’ (5’ phosphate to 3’ hydroxyl)
Synthesis is the same as the leading strand of DNA

22. RNA Polymerase
RNA is released so we can make many copies of the gene, usually before the first one is done
Can have multiple RNA polymerase molecules on a gene at a time

23. Differences in DNA and RNA Polymerases
RNA polymerase adds ribonucleotides not deoxynucleotides
RNA polymerase does not have the ability to proofread what they transcribe
RNA polymerase can work without a primer
RNA will have an error 1 in every 10,000 nucleotides (DNA is 1 in 10,000,000 nucleotides)

24. Types of RNA messenger RNA (mRNA) – codes for proteins
ribosomal RNA (rRNA) – forms the core of the ribosomes, machinery for making proteins
transfer RNA (tRNA) – carries the amino acid for the growing protein chain

25. DNA Transcription in Bacteria
RNA polymerase must know where the start of a gene is in order to copy it
RNA polymerase has weak interactions with the DNA unless it encounters a promoter
A promoter is a specific sequence of nucleotides that indicate the start site for RNA synthesis

26. RNA Synthesis
RNA pol opens the DNA double helix and creates the template
RNA pol moves nt by nt, unwinds the DNA as it goes
Will stop when it encounters a STOP codon, RNA pol leaves, releasing the RNA strand

27. Sigma () Factor
Part of the bacterial RNA polymerase that helps it recognize the promoter
Released after about 10 nucleotides of RNA are linked together
Rejoins with a released RNA polymerase to look for a new promoter

28. Start and Stop Sequences

29. DNA Transcribed
The strand of DNA transcribed is dependent on which strand the promoter is on
Once RNA polymerase is bound to promoter, no option but to transcribe the appropriate DNA strand
Genes may be adjacent to one another or on opposite strands

30. Eukaryotic Transcription
Transcription occurs in the nucleus in eukaryotes, nucleoid in bacteria
Translation occurs on ribosomes in the cytoplasm
mRNA is transported out of nucleus through the nuclear pores

31. RNA Processing
Eukaryotic cells process the RNA in the nucleus before it is moved to the cytoplasm for protein synthesis
The RNA that is the direct copy of the DNA is the primary transcript
2 methods used to process primary transcripts to increase the stability of mRNA being exported to the cytoplasm
RNA capping
Polyadenylation

32. RNA Processing
RNA capping happens at the 5’ end of the RNA, usually adds a methylgaunosine shortly after RNA polymerase makes the 5’ end of the primary transcript
Polyadenylation modifies the 3’ end of the primary transcript by the addition of a string of A’s

33. Coding and Non-coding Sequences
In bacteria, the RNA made is translated to a protein
In eukaryotic cells, the primary transcript is made of coding sequences called exons and non-coding sequences called introns
It is the exons that make up the mRNA that gets translated to a protein

34. RNA Splicing
Responsible for the removal of the introns to create the mRNA
Introns contain sequences that act as cues for their removal
Carried out by small nuclear riboprotein particles (snRNPs)

35. snRNPs
snRNPs come together and cut out the intron and rejoin the ends of the RNA
Intron is removed as a lariat – loop of RNA like a cowboy rope

36. Benefits of Splicing Allows for genetic recombination
Link exons from different genes together to create a new mRNA
Also allows for 1 primary transcript to encode for multiple proteins by rearrangement of the exons

37. Summary

38. RNA to Protein Translation is the process of turning mRNA into protein
Translate from one “language” (mRNA nucleotides) to a second “language” (amino acids)
Genetic code – nucleotide sequence that is translated to amino acids of the protein

39. Degenerate DNA Code
Nucleotides read 3 at a time meaning that there are 64 combinations for a codon (set of 3 nucleotides)
Only 20 amino acids
More than 1 codon per AA – degenerate code with the exception of Met and Trp (least abundant AAs in proteins)

40. Reading Frames
Translation can occur in 1 of 3 possible reading frames, dependent on where decoding starts in the mRNA

41. Transfer RNA Molecules
Translation requires an adaptor molecule that recognizes the codon on mRNA and at a distant site carries the appropriate amino acid
Intra-strand base pairing allows for this characteristic shape
Anticodon is opposite from where the amino acid is attached

42. Wobble Base Pairing
Due to degenerate code for amino acids some tRNA can recognize several codons because the 3rd spot can wobble or be mismatched
Allows for there only being 31 tRNA for the 61 codons

43. Attachment of AA to tRNA
Aminoacyl-tRNA synthase is the enzyme responsible for linking the amino acid to the tRNA
A specific enzyme for each amino acid and not for the tRNA

44. 2 ‘Adaptors’ Translate Genetic Code to Protein1
 2

45. Ribosomes Complex machinery that controls protein synthesis 2 subunits
1 large – catalyzes the peptide bond formation
1 small – binds mRNA and tRNA
Contains protein and RNA
rRNA central to the catalytic activity
Folded structure is highly conserved
Protein has less homology and may not be as important

46. Ribosome Structures May be free in cytoplasm or attached to the ER
Subunits made in the nucleus in the nucleolus and transported to the cytoplasm

47. Ribosomal Subunits
1 large subunit – catalyzes the formation of the peptide bond
1 small subunit – matches the tRNA to the mRNA
Moves along the mRNA adding amino acids to growing protein chain

48. Ribosomal Movement 4 binding sites E-site mRNA binding site
Peptidyl-tRNA binding site (P-site)
Holds tRNA attached to growing end of the peptide
Aminoacyl-tRNA binding site (A-site)
Holds the incoming AA
Exit site (E-site)

49. 3 Step Elongation Phase Elongation is a cycle of events
Step 1 – aminoacyl-tRNA comes into empty A-site next to the occupied P-site; pairs with the codon
Step 2 – C’ end of peptide chain uncouples from tRNA in P-site and links to AA in A-site
Peptidyl transferase responsible for bond formation
Each AA added carries the energy for the addition of the next AA
Step 3 – peptidyl-tRNA moves to the P-site; requires hydrolysis of GTP
tRNA released back to the cytoplasmic pool

50. Initiation Process
Determines whether mRNA is synthesized and sets the reading frame that is used to make the protein
Initiation process brings the ribosomal subunits together at the site where the peptide should begin
Initiator tRNA brings in Met
Initiator tRNA is different than the tRNA that adds other Met

51. Ribosomal Assembly Initiation Phase
Initiation factors (IFs) catalyze the steps – not well defined
Step 1 – small ribosomal subunit with the IF finds the start codon –AUG
Moves 5’ to 3’ on mRNA
Initiator tRNA brings in the 1st AA which is always Met and then can bind the mRNA
Step 2 – IF leaves and then large subunit can bind – protein synthesis continues
Met is at the start of every protein until post-translational modification takes place

52. Eukaryotic vs Procaryotic
No CAP; have specific ribosome binding site upstream of AUG
Polycistronic – multiple proteins from same mRNA
Eucaryotic
Monocistronic – one polypeptide per mRNA

53. Protein Release Protein released when a STOP codon is encountered
UAG, UAA, UGA (must know these sequences!)
Cytoplasmic release factors bind to the stop codon that gets to the A-site; alters the peptidyl transferase and adds H2O instead of an AA
Protein released and the ribosome breaks into the 2 subunits to move on to another mRNA

54. Polyribosomes
As the ribosome moves down the mRNA, it allows for the addition of another ribosome and the start of another protein
Each mRNA has multiple ribosomes attached, polyribosome or polysome

55. Regulation of Protein Synthesis
Lifespan of proteins vary, need method to remove old or damaged proteins
Enzymes that degrade proteins are called proteases – process is called proteolysis
In the cytosol there are large complexes of proteolytic enzymes that remove damaged proteins
Ubiquitin, small protein, is added as a tag for disposal of protein

56. Protein Synthesis
Protein synthesis takes the most energy input of all the biosynthetic pathways
4 high-energy bonds required for each AA addition
2 in charging the tRNA (adding AA)
2 in ribosomal activities (step 1 and step 3 of elongation phase)

57. Summary

58. Ribozyme
A RNA molecule can fold due to its single stranded nature and in folding can cause the cleavage of other RNA molecules
A RNA molecule that functions like an enzyme hence ribozyme name