Transcript Transcription Translation

From Gene to Protein
PROTEIN SYNTHESIS
You Must Know
 The key terms :
 Transcription and translation
 Explain the process of transcription
 How eukaryotic cells modify RNA after transcription
 The steps of translation
 How point mutations change the amino acid
sequence of a protein
Hint from me as a reader
 Central Chapter for Molecular
Genetics- One of the top 5 to study
for AP EXAM
Genes Specify
Proteins
Via Transcription and
Translation
One Gene one
polypeptide
hypothesis
Each
gene codes for a
polypeptide which can
be or be part of a
protein
Overview of Transcription/Translation
Transcription
Translation
 Synthesis of RNA or mRNA
 Polypeptide production of
under the direction of DNA
 mRNA is produced
 Prem-RNA undergoes
processing to yield mRNA
 Prokaryotes- no premRNA
polypeptide chain using mRNA
transcript that occurs in
ribosome
 Uses triplet code-codon
 Single strand DNA transcribed
to complementary to the
original DNA
 mRNA base triplet codes are
called codons, 5’-3’ direction

Transcription and translation
simultaneously produced
Redundant Genetic Code
 More than 1 codon codes for each of the 20
amino acids
 Codons are in groups of 3 and are read in a
frame
 Must be read in correct groupings for
translation to be successful
Prove it!!!
 Archibald Garrod



First to suggest genes
dictate phenotype through
enzymes
Inborn errors of
metabolism
May be the first to
recognize Mendel’s pea
characteristics of
inheritance apply to
humans as well
More Proof!!!
 Beadle and Tatum




Bread mold (Neurospora)
Bombarded with x-rays to
mutate
Medium (agar) had 20
amino acids to
supplement mutant’s
growth even if 1 was
missing.
Plated these on specific
medium to determine
what supplements were
missing from fungi
Nutritional Mutants in fungi
 Beadle and Tatum





Bread mold exposed to xrays
Mutants that were unable
to survive on minimal
media
Found 3 classes of
arginine deficient mutants
Each lacked a different
enzyme for catalysis
Developed One Gene one
Enzyme Hypothesis
Central Dogma
Universality of the genetic code
 Nearly universal from
simple bacteria to
complex organisms
 Transcription and
translation can occur in
genes transplanted from
one species to another
Transcription
 Defined as the DNA
directed synthesis of
RNA

STEP 1

RNA polymerase separates
the 2 DNA strands and
connects RNA nucleotides
as they base pair along the
DNA template forms
mRNA specifically premRNA
Addition of RNA nucleotides
 2. RNA polymerase add
RNA only in the 3’ end,
elongates in the 5’-3’ end
 Remember uracil
replaces thymine
 First attachment is called
the Promoter, and yes, it
is called a TATA box
which binds first to the
DNA before RNA
polymerase II can call
transcription unit
 Terminator-end
Synthesis of RNA transcript- stage 1Initiation
Bacteria
Eukaryotes
 RNA polymerase to the
 RNA polymerase II
promoter
cannot bind or attach
without proteins called
transcription factors- all
called transcription
initiation complex
 Crucial DNA Promoter is
TATA that forms the
complex
 RNA unwinds DNA
Transcription Initiation Complex
2. Elongation of the RNA strand
 RNA polymerase moves
along DNA
 Untwists helix
 RNA nucleotides added
to 3’end
 Double helix reforms
leaving hanging RNA
strand
3. Termination
 After RNA polymerase
hits a terminator
sequence in DNA stops
 RNA transcript released
 Polymerase detaches
Are we done yet?
 No!!! Modification of
RNA after transcription
 ADDITION OF





Poly-A tail
5’cap
Helps with export from
nucleus
Protection from
degradation of enzymes
Attachment to ribosomes
RNA Splicing of pre-mRNA
 Eukaryotic cells
 Introns are sections cut
out
 Parts that are kept are
exons
 Spliceosome



do the splicing
RNA or snRNA (small
nuclear RNA)
Ribozyme- RNA that acts
as an enzyme
Editing of premRNA
 intron and Exon Splicing
What is a snRNPs?
 Particles called small
nuclear
ribonucleoproteins
(called snurps) are small
nuclear RNA plus a
protein are called snRNP
and these recognize
splice sites
 Different snurps +
additional protein form
spliceosome
Introns
 Have functional and
evolutionary importance




May regulate gene
expression
Genes can encode more
than 1 kind of polypeptide
depending on which
segments are exons
(alternative RNA splicing)
Number of proteins
(100,000)produced
exceeds number of
genes(25,000)
Translation
 RNA directed synthesis
of a polypeptide in the
cytoplasm on the
ribosome

t-RNA


Transfers correct t-RNA
from pool in the cytoplasm
R-RNA

Accepts aa from t-RNA
and binds aa at the other
end with a triplet called
anticodon
Structure and function of tRNA
 Molecules of tRNA

Not identical
Each carries specific aa on
one end
 Anticodon on one end
base-pairs with the
complementary codon on
mRNA
 80 nucleotides long
 Flattened into one plane,
cloverleaf shape
 H bonds cause tRNA twist
 Roughly L-shaped

Structure and Function of rRNA
 Complexes Proteins and
tRNA

3 binding sites
P-holds tRNA growing
polypeptide chain
 A- holds tRNA that carries
the amino acids added to
next chain
 E-exit site for tRNA

Ribosomes
 Facilitate coupling of tRNA anticodon to mRNA
codons during protein synthesis
 Ribosomal subunits are made of proteins and
ribosomal RNA (rRNA)
 Bacteria and Eukaryotic ribosomes are somewhat
similar but have significant differences

Antibiotic drugs specifically target bacterial ribosomes without
harming eukaryotic ribosomes
Figure 17.17
Growing
polypeptide
tRNA
molecules
E P
Exit tunnel
Large
subunit
A
Small
subunit
5
mRNA
3
(a) Computer model of functioning ribosome
Growing polypeptide
P site (Peptidyl-tRNA
binding site)
Exit tunnel
Next amino
acid to be
added to
polypeptide
chain
A site (AminoacyltRNA binding site)
E site
(Exit site)
E
mRNA
binding site
Amino end
P
A
Large
subunit
Small
subunit
(b) Schematic model showing binding sites
E
tRNA
mRNA
5
3
Codons
(c) Schematic model with mRNA and tRNA
3 Stages of Translation
 Initiation

Has 3 steps
 Elongation

Has 3 steps
 Termination

Stop codon in the mRNA
is reached and translation
stopped
Initiation steps
 1. mRNA with the code
AUG is in the proper
position
 2. tRNA anticodon with
UAC (methionine)
hydrogen bonds to first
with initiation factors
assist in holding together
 3. Subunit of ribosome
allows methionine to
attach to the P site
Elongation Steps
 1. Codon recognition



Enters the A site
Anticodon of incoming
aminoacyl-tRNA base
pairs withcomplement on
A site
GTP hydrolysis insures
accuracy and efficiency
Figure 17.16-1
Amino acid
P P P Adenosine
ATP
Aminoacyl-tRNA
synthetase (enzyme)
Figure 17.16-2
Aminoacyl-tRNA
synthetase (enzyme)
Amino acid
P Adenosine
P P P Adenosine
ATP
P Pi
Pi
Pi
Figure 17.16-3
Aminoacyl-tRNA
synthetase (enzyme)
Amino acid
P Adenosine
P P P Adenosine
P Pi
ATP
Pi
Pi
tRNA
Aminoacyl-tRNA
synthetase
tRNA
Amino
acid
P Adenosine
AMP
Computer model
Figure 17.16-4
Aminoacyl-tRNA
synthetase (enzyme)
Amino acid
P Adenosine
P P P Adenosine
P Pi
ATP
Pi
Pi
tRNA
Aminoacyl-tRNA
synthetase
tRNA
Amino
acid
P Adenosine
AMP
Computer model
Aminoacyl tRNA
(“charged tRNA”)
Elongation Steps
 2. Peptide bond
formation on P site


rRNA catalyzes the
formation of a peptide
bond on the new amino
acid to the carboxyl end of
the growing polypeptide
on the P site
Removes the polypeptide
from the A site to the P
site
Elongation Steps
 3. Translocation

Ribosomes translocates
the tRNA in the A site to
the P site, empty tRNA on
the P site is moved to the
E site where it is released
Video of the Process
 http://www.hhmi.org/biointeractive/translation-
basic-detail
Termination
 A termination stop codon
in the mRNA is reached


Release factor (protein)
binds to the stop codon
Polypeptide is freed
Wobble
 Is an mRNA triplet
 64 different codons
 mRNA reads codon by
codon, 1 aa added to the
chain
 Relaxation of reading the
3rd base on t-RNA
Accurate Translation
 1. Correct match b/t
tRNA and an amino acid

Aminoacyl-tRNA
synthetase (enzyme)
 Anticodon and mRNA
codon match
 WOBBLE


Flexible pairing at the 3rd
base of a codon
tRNA bind to more than
one codon
Completing and Targeting functional protein
 Translation is not
 Synthesis begins and
enough
 Chains are modified
 Targeted to specific sites
in the cell
ends in cytosol unless the
polypeptide signals the
ribosome to attach to the
ER
 Those destined for ER or
secretion marked by
signal peptide-Signal
recognition particle
(SRP) which brings the
signal peptide to the ER

Bound ribosomes


Endomembrane-secreted
out
Free ribosomes-cytosol
 Protein folding
 Post-translational
modifications
Polyribosome
 Ribosomes can translate a single mRNA
simultaneously


Forms polyribosome or polysome
Multiple copies of polypeptide quickly
Figure 17.21
Growing
polypeptides
Completed
polypeptide
Incoming
ribosomal
subunits
Start of
mRNA
(5 end)
(a)
End of
mRNA
(3 end)
Ribosomes
mRNA
(b)
0.1 m
Figure 17.21a
Ribosomes
mRNA
0.1 m
Properties of RNA
 3 properties of RNA



1.forms 3 dimensional
structure can base-pair to
itself
2. Some bases in RNA
contain functional groups
that may participate in
catalysis
3. RNA can H bond to
other nucleic acid
molecules
Figure 17.22
1 Ribosome
5
4
mRNA
Signal
peptide
3
SRP
2
ER
LUMEN
SRP
receptor
protein
Translocation
complex
Signal
peptide
removed
ER
membrane
Protein
6
CYTOSOL
Mutations
 Affect protein structure and function
 Changes in the genetic material of the cell or virus
 Substitutions
Nucleotide –pair substitution
 Silent mutation- no affect due to redundancy
 Missense-codes for incorrect aa
 Nonsense-codes to a stop codon and leads to nonfunctional


Point mutations
Single base
 Leads to the production of an abnormal protein
 Nucleotide –pair substitution
 Insertions and deletions

Mutations
 Insertions and
Delections



Additions or losses of
nucleotide pairs
Disastrous effect on the
resulting protein
May alter the reading
frame to a frameshift
mutation
Progeria
 Error LMNA gene, a
protein that provides
support to the cell
nucleus.
 Accelerated aging
Errors
Insertions/deletions
Mutagens
 Spontaneous mutations
during DNA replication,
recombination or repair
 Physical or chemical
agents that cause
mutations




X-rays, UV light
Aspertame
Saccharin
Polycyclic aromatic
hydrocarbons- smoked
meat and fish
Bacteria, eukarya, Archaea
Bacteria
Eukarya
Archaea
Simultaneously transcribe Transcription and
and translate the same
translation are separated
gene
by the nuclear envelope
Likely coupled of
transcription and
translation
Proteins diffuse to sites
Same size ribosomes as
bacteria but function is
different
No transcription sitesaccessory proteins
Resemble eukaryotes in
RNA polymerase,
termination of
transcription and
ribosomes
Figure 17.26
TRANSCRIPTION
DNA
3
5
RNA
polymerase
RNA
transcript
Exon
RNA
PROCESSING
RNA transcript
(pre-mRNA)
AminoacyltRNA synthetase
Intron
NUCLEUS
Amino
acid
AMINO ACID
ACTIVATION
tRNA
CYTOPLASM
mRNA
Growing
polypeptide
3
A
Aminoacyl
(charged)
tRNA
P
E
Ribosomal
subunits
TRANSLATION
E
A
Anticodon
Codon
Ribosome