Transcription and Translation

The Relationship
Between Genes and
Proteins
- Translation -
From gene to protein: genetic code
 Information travels from DNA to RNA to Protein
• Is there a one-to-one correspondence between DNA,
RNA and Protein?
– DNA and RNA each have four nucleotides that can form them; so
yes, there is a one-to-one correspondence between DNA and
RNA.
– Proteins can be composed of a potential 20 amino acids; only four
RNA nucleotides: no one-to-one correspondence.
– How then does RNA direct the order and number of amino acids in
a protein?
• How many bases are required for each amino acid?
• (4 bases)2bases/aa = 16 amino acids—not enough
• (4 bases)3bases/aa = 64 amino acid possibilities
• Minimum of 3 bases/aa required
From gene to protein: genetic code
• Nirenberg and Matthaei Experiment:
 Found that adding rRNA prolonged cell-free protein
synthesis
 Adding artificial RNA synthesized by polynucleotide
phosphorylase (no template, UUUUUUUUU)
stimulated protein synthesis more
 The protein that came out of this reaction was
polyphenylalanine (UUU = Phe)
 Other artificial RNAs: AAA = Lys; CCC =Pro
 Nirenberg and Matthaei Experiment
1) They set up twenty test tubes,
each with a different (radioactively
-labeled) amino acid.

2) E.coli bacterial cytoplasm was

added to each test tube.
(Ribosomes etc. from bacteria)

3) Artificially synthesized mRNA with the codon UUU only,

was place in each test tube.

4) Then analyzed each test tube to find out which one

contains amino acids bonded in a chain.
Nirenberg and Matthaei
Experiment
 Nirenberg and Matthaei Experiment
 They looked for the protein produced with the radiation.
It was indeed made up of just phenylalanine.
 Therefore Nirenberg and Matthaei knew the codon UUU
represented the amino acid phenylalanine.
 They did a similar experiment with
AAA which represents Lysine. They
then published their results.
 Nirenberg assembled a team of
scientists to crack the rest of the
genetic code.
From gene to protein: genetic code
• Nirenberg:
 Triplet binding assay: add triplet RNA, ribosomes,
binding factors, GTP, and radiolabeled charged tRNA.
• UUU trinucleotide binds to Phe-tRNA
• UGU trinucleotide binds to CYS-tRNA
 By fits and starts the triplet genetic code was worked
out.
 Each three-letter “word” (codon) specifies an amino
acid or directions to stop translation.
 The code is redundant or degenerate: more than one
way to encode an amino acid
 Genetic Code: Triple Codons
• Found that 61 of 64 possible codons
code for an amino acid
• Notice the redundancy (degeneracy)
- UCU, UCC, UCA, UCG, AGU, AGC all
code for serine
- Third position often unimportant (UC_)
- H bonding is more important in 1st 2
positions
• Not all codons code for an amino acid
- 3 stop codons exist
- When ribosome reads,
termination occurs and protein
synthesis ceases.
- AUG is always the start codon
- Thus, all proteins start with a
methionine
Genetic Code: Triple Codons
From gene to protein: Translation
• Components required for translation:
 mRNA: resulting from transcription
 Ribosomes: a complex macromolecular machine that serves as the
site of biological protein synthesis (translation). Ribosomes link amino
acids together in the order specified by messenger RNA (mRNA)
molecules.
 tRNA: transfer ribonucleic acid (tRNA) is a type of RNA molecule
that helps decode a messenger RNA (mRNA) sequence into a
protein.
 Aminoacyl tRNA synthetases: is an enzyme that catalyzing the
esterification of a specific amino acid to its compatible tRNAs to form
an aminoacyl-tRNA.
 Initiation, elongation and termination factors: are a set of
proteins that are used in protein synthesis in the process of initiation,
elongation and termination.
Translation: Ribosomes
• Translation – Process by which a ribosome reads the
sequence of codons in a strand of mRNA and uses the
information to produce a polypeptide from amino acids
• Ribosomes are made up of 2 subunits – a small and a large
- Each subunit contains 20-50 different proteins and
several molecules of ribsomal RNA (rRNA)
- Differences in sizes exist between prokaryotic and
eukaryotic ribosomes
1). Prokaryotic – 70S
- Large (50S) + small (30S)
- Contains 16S rRNA
- Used for species ID
2). Eukaryotic – 80S
- Large (60S) + small (40S)
Ribosomal Proteins
 Translation: Ribosomes
• All ribosomes have 1 mRNA binding site and 3 binding sites
for tRNAs
- Remember tRNAs are going to bring the appropriate amino
acids to the ribosome.
- A (aminoacyl) site = Holds a tRNA
that just arrives to the ribosome
- P (peptidyl) site = Holds a
tRNA that contains the
growing polypeptide chain
- E (exit) site = Holds a tRNA that
has already given up its amino
acid and is getting ready to exit
the ribosome
Translation: The Process
Translation can be divided into 3 major stages:
1). Initiation – Getting the ribosome ready
2). Elongation – Stage when the polypeptide is actually
made
3). Termination – The process stops
Translation: Initiation (at glance)
Initiation – Getting the ribosome ready
• Ribosome small subunit binds to mRNA
• Charged tRNA anticodon forms base pairs with the mRNA
codon
• Small subunit interacts with initiation factors and special
initiator tRNA that is charged with methionine
• mRNA-small subunit-tRNA complex recruits the large
subunit
• Eukaryotic and prokaryotic initiation differ slightly
• The large subunit of ribosome contains three binding sites:
 Amino acyl (A site)
 Peptidyl (P site)
 Exit (E site)
• At initiation,
 The tRNAfMet occupies the P site
 A second, charged tRNA complementary to the next codon binds
the A site.
Translation: Initiation (more detail)
Initiation – Getting the ribosome ready
a).Several initiation factors bind to the small ribosomal subunit
- Initiation factors (IFs) are proteins that help get the ribosome
/mRNA/tRNA assembled
- IF3 binds to the small subunit and helps keep it apart
from the large subunit
b). Recruitment of an initiator tRNA to the small subunit
- IF2 (euk and prok) binds to an initiator tRNA and brings it to
the P site of the small subunit
- Initiator tRNA contains the anticodon UAC and a the
amino acid methionine
- Why this tRNA?
- Remember the universal start codon is AUG
- Thus need to start with a tRNA that has the anticodon UAC
Translation: Initiation
 Initiation factors IF-3, initiator tRNA with N-
formylmethionine attached to form fmet-tRNA
 Energy molecule: GTP
 The Shine–Dalgarno consensus sequence in bacterial
cells is recognized by the small unit of ribosome.

 The Kozak sequence in eukaryotic cells facilitates the

identification of the start codon.
Translation: Initiation
c) The mRNA binds to the small ribosomal subunit
 In eukaryotic cells, a complex of initiation factors
(collectively called IF4) binds to the mRNA 5' cap
 IF4 on the mRNA interacts with IF3, which is already
bound to the small subunit
 Starting at the end, the small subunit begins scanning
the mRNA until reaching the first available AUG (start
codon)
 Ribosome helps identify the start codon by the presence
of an upstream Kozak sequence

AUG AUG

- Bacterial mRNA don't have a cap – bind to ribosome via IF3 only (no
scanning from the end).
- Shine-Dalgarno sequence before AUG helps the ribosome find it
Translation: Initiation
d) The large subunit binds to the assembled complex
- The large ribosomal subunit comes in and attaches to the
small subunit complex
- Most of the other initiation factors then come off the complex
 The ribosome is now ready to begin constructing a
polypeptide
Translation: Initiation
Translation: elongation (KEYWORD)
• Exit (E-site): is the third and final binding site for t-RNA in
the ribosome during protein synthesis. The "E" stands for
exit, and is accompanied by the P-site (for peptidyl) which is
the second binding site, and the A-site (aminoacyl), which is
the first binding site. It is involved in cellular processes
• Peptidyl (P-site): is the second binding site for tRNA in
the ribosome. During protein translation, the P-site holds the
tRNA which is linked to the growing polypeptide chain.
• Aminoacyl (A-site): is the first binding site for charged 
t-RNA molecules during protein synthesis.
• Elongation factors (Tu, Ts, and G): are a set of
proteins that facilitate process of elongation.
Translation: elongation (at glance)
Elongation – Stage when the polypeptide is actually made
• Ribosome translocates by three bases after peptide bond
formed
• New charged tRNA aligns in the A site
• Peptide bond between amino acids in A and P sites is
formed
• Ribosome translocates by three more bases
• The uncharged tRNA in the A site is moved to the E site.
 EF-Tu recruits charged tRNA to A site. Requires hydrolysis
of GTP
 Peptidyl transferase catalyzes peptide bond formation (bond
between aa and tRNA in the P site converted to peptide
bond between the two amino acids)
 Peptide bond formation requires RNA and may be a
ribozyme-catalyzed reaction
Translation: elongation (more detail)
2) Elongation – Stage when the polypeptide is actually
made
a) The ribosome reads the next codon (3 nucleotides) and the
specific tRNA with the complimentary anticodon will come into
the A site
- Attachment of tRNAs to the ribosome
is aided by various elongation factors
(EFs) in prokaryotes and eukaryotes

b)The methionine is removed from the

1st tRNA and enzymatically added to
the amino acid on the 2nd tRNA (the
one in the A site)
- A peptide bond is formed
- This transfer reaction is catalyzed
by the rRNA in the ribosome
Translation: elongation
c)The empty (uncharged) tRNA in the P
site moves into the E site briefly and
exits
the ribosome
- tRNA movement is aided by other
EFs

d) mRNA shifts by 3 nucleotides (also

aided by EFs)
- The tRNA with the 2 amino acids
moves to the P site
- The 3rd codon is now exposed in the
A site

e) The tRNA with the complimentary

anticodon comes into the A site
 Translation: elongation
f) Dipeptide on the tRNA in the P site is transferred onto the
amino acid in the A site............
 The whole process continues – a polypeptide chain is created
 The order of codons determines the order of amino acids!!
Translation: termination (KEYWORD)
• Termination/STOP codons (UAA, UAG, and UGA): is
a nucleotide triplet within messenger RNA that signals a
termination of translation into proteins.

• Release factors (Rf): is a protein that allows for the

termination of translation by recognizing the 
termination codon or stop codon in an mRNA sequence.
Translation: Termination (at glance)
• Elongation proceeds until STOP codon reached
 UAA, UAG, UGA
• No tRNA normally exists that can form base pairing with a
STOP codon; recognized by a release factor
• tRNA charged with last amino acid will remain at P site
• Protein release factors bind to the ribosome and release both
mRNA and polypeptide (cut polypeptide from last tRNA)
• Ribosome subunits dissociate from each other
 Review of Translation

Animation: http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter15/animations.html