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
4. Central Dogma
• 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
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. 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
9. Transcription
• Similarities to DNA replication
– 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
10. Template to Transcripts
• The RNA transcript is identical to the NON-
template strand with the exception of the T’s
becoming U’s
11. 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
12. 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
13. 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
14. 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)
15. 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
16. 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
17. 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
18. 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
20. 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
21. 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
22. 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
23. 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
24. 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
25. 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)
26. 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
27. 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
29. 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
30. 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)
31. Reading Frames
• Translation can occur in 1 of 3 possible reading
frames, dependent on where decoding starts in the
mRNA
32. 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
33. 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
34. 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
36. 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
37. 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
38. 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
39. Ribosomal Movement
• 4 binding sites
– 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)
E-site
40. 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
41. 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
42. 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
43. Eukaryotic vs Procaryotic
• Procaryotic
– No CAP; have specific ribosome binding site upstream of AUG
– Polycistronic – multiple proteins from same mRNA
• Eucaryotic
– Monocistronic – one polypeptide per mRNA
44. 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
45. 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
46. 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
47. 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)
49. 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
