Download Chapter 17 * from gene to protein

CHAPTER 17 – FROM
GENE TO PROTEIN
The information content of genes is in the form
of specific sequences of nucleotides along the
DNA strands. The DNA of an organism leads to
specific traits by dictating the synthesis of
proteins and of RNA molecules involved in
protein synthesis (gene expression.)
Proteins are the link between genotype and
phenotype.
1
ARCHIBALD GARROD
The study of metabolic defects provided evidence that
genes specify proteins.
Garrod discovered that proteins (enzymes) are the link
between genotype and phenotype.
He figured out that some inherited diseases are the
inability to make enzymes
He noticed that the diaper of a baby was very brown.
He determined that the baby had alkaptonuria,
which is a recessively inherited disorder where the
urine is a brown color. This is due to homogentisic
acid which cannot be broken down in the body, so it
is excreted in the urine. The reason it cannot be
broken down is because there is an absence of the
enzyme needed in the biochemical pathway.
2
Beadle and Tatum showed the relationship between genes and
enzymes. They used the bread mold Neurospora and exposed it
to X-rays to get mutants. They found 3 different classes of
mutants. Each mutant was defective in a different gene. They
exposed these mutants to different environments to see which
ones allowed arginine to grow. They deduced that each mutant
was unable to carry out one step in the arginine pathway –
probably because it lacked the necessary enzyme
BEADLE
AND TATUM
3
ONE GENE – ONE ENZYME HYPOTHESIS…AND
THE EVOLUTION OF THAT HYPOTHESIS
From Beadle and Tatums experiments, they came up with the one gene, one
enzyme hypothesis.
However, not all proteins are enzymes, so it became the one gene- one protein
hypothesis.
BUT…some genes have more than one polypeptide (THINK: quaternary
structure of proteins), so it led to the one gene- one polypeptide hypothesis.
The newest discoveries have been taken into consideration and the scientific
community have updated the definition of a gene as:
A gene is a region of DNA that can be expressed to produce a final functional
product that is either a polypeptide or an RNA molecule.
4
OVERVIEW: TRANSCRIPTION
TRANSLATION
DNA
transcription
Primary transcript (pre-mRNA)
RNA processing
mRNA
translation
protein
Genes provide the instructions for making
specific proteins and getting from gene to protein
needs two stages:
Transcription = DNA → RNA
Translation = RNA → Protein
5
TRANSCRIPTION AND TRANSLATION IN
PROKARYOTES VS. EUKARYOTES



The basic mechanics of
transcription and
translation are similar in
eukaryotes and bacteria.
Bacteria lack nuclei, and
their DNA is not
separated from
ribosomes and other
protein-synthesizing
equipment.
 This allows the
coupling of
transcription and
translation.
In a eukaryotic cell,
transcription occurs in the
nucleus, and translation
occurs at ribosomes in
the cytoplasm.
The molecular chain of command in a cell has a
directional flow of genetic information:
DNA  RNA  protein
Francis Crick dubbed this concept the central dogma
in 1956.
6
CODING FOR
AMINO ACIDS
The message is carried in
RNA in the form of codons
(3 bases). It is read in the
5’ → 3’ direction.
7





With a triplet code, three
consecutive bases specify an
amino acid, creating 43 (64)
possible code words.
During transcription, one DNA
strand, the template strand,
provides a template for
ordering the sequence of
nucleotide bases in an mRNA
transcript.
The mRNA base triplets are
called codons.
Each codon specifies which
one of the 20 amino acids will
be incorporated at the
corresponding position along a
polypeptide chain.
The starting point establishes
the reading frame; subsequent
codons are read in groups of
three nucleotides.
THE TRIPLETS
(CODONS) CODE
FOR THE SPECIFIC
AMINO ACIDS
8
The genetic code must
have evolved very early
in the history of life It
is nearly universal,
shared by organisms
from the simplest
bacteria to the most
complex plants and
animals.
Nirenberg determined the first match: UUU
codes for the amino acid phenylalanine.
Sixty-one of 64 triplets code for amino
acids.
Marshall Nirenberg
deciphered the code for
the amino acids in 1961.
The codon AUG not only codes for the
amino acid methionine but also indicates
the “start” or initiation of translation.
Three codons do not indicate amino acids
but are “stop” signals marking the
termination of translation.
9
TRANSCRIPTION
DNA → RNA
Promoter – DNA sequence where
RNA attaches and initiates
transcription
Terminator – sequence that signals
the end of transcription
Transcription Unit – sequence of
DNA that is transcribed into RNA
3 Steps of Transcription:
1. Initiation
2. Elongation
3. Termination
10
TRANSCRIPTION INITIATION
The promoter determines which
strand is the template and then
transcription factors help RNA
polymerase bind. The TATA box is an
important part of the promoter that
helps initiate transcription. The
transcription complex consists of the
promoter, transcription factors, and
RNA polymerase.
RNA polymerase separates the DNA
strands at the appropriate point and
joins RNA nucleotides
complementary to the DNA template
strand. Like DNA polymerases, RNA
polymerases can assemble a
polynucleotide only in its 53
direction (therefore the template
strand is 3’  5’.)
11
TRANSCRIPTION ELONGATION
The RNA polymerase adds RNA
nucleotides about 10-20 at a
time to the growing 3’ end.
Several mRNA strands can be
made at the same time….several
different RNA polymerases can
all be on the same DNA molecule
and can all create mRNA. This
helps the cell make the encoded
protein in large amounts.
12
TRANSCRIPTION TERMINATION
In prokaryotes, termination stops at
the termination signal (end of the
gene)
In eukaryotes, transcription continues
for 10-35 nucleotides past the stop
signal. Later in the process, it gets cut
down.
At this point, transcription has given us the primary transcripts or pre-mRNA
13
RNA PROCESSING:
MODIFYING THE PRE-MRNA
- At the 5’ end, a 5’ cap is added (which is a modified guanine molecule)
- At the 3’ end, there is the poly-A tail (50-250 adenine nucleotides)  functions in
helping to inhibit degradation and helps exportation from nucleus)
-- Both of these modifications have several important functions:
-Exporting mRNA from the nucleus
-Protecting mRNA from hydrolytic enzymes
-Helping the ribosome attach to the 5’ end of the mRNA
After both ends are
modified, the introns
(non-coding portions)
are spliced out.
14
Introns = noncoding
segments
Exons = coding
segments
RNA SPLICING
The introns are cut out using
splicesomes. Therefore, the
mRNA that leaves the nucleus
(exons only) is the abridged
version that only carries genes –
not “filler” DNA.
15
RNA SPLICING
TECHNIQUE SPLICESOMES
There are short sequences at the
end of introns that signal to the
snRNP’s (small nuclear
ribonucleoproteins). The snRNP’s
recognize these sites and the
splicesomes then cut out the
introns and reattach the exons.
Ribozyme → RNA molecules that
act like enzymes; in some
organsisms RNA splicing can occur
without additional proteins
because the introns can catalyze
their own excision
16
ALTERNATIVE RNA SPLICING
Humans can get along with a small number
of genes because we can “shuffle” our DNA;
different polypeptides can be made
depending on which segments we consider
introns and which are considered exons.
17
TRANSLATION – FROM RNA TO PROTEIN
BUILDING A
POLYPEPTIDE!
18
tRNA - TRANSLATOR
-The cell is always stocked
with all 20 AA’s (from diet)
-The tRNA is folded like a
cloverleaf; on one side it
has an anticodon that
matches up with the
codon from the mRNA; on
the other side it carries a
specific AA
-The tRNA’s are used over
and over; they drop off
their AA’s and then go get
another to be used again
Wobble → relaxation of 3rd base pairing; sometimes the 3rd base of the ANTICODON has an
“I” (inosine), which is an altered adenine; this can match up with U, C, or A; If each
anticodon had to be a perfect match to each codon, we would expect to find 61 types of
tRNA, but the actual number is about 45, because the anticodons of some tRNAs recognize
more than one codon (the wobble!!)
CCI anticodon can match up with GGU, GGC
19
and GGA (codons)
AMINOACYL-tRNA
SYNTHETASE
This enzyme attaches each AA to its
appropriate tRNA.
This process uses 1 ATP
There are 20 different aminoacyl-tRNA
synthetases (one for each AA)
Process:
1. The active site of the aminoacyl tRNA
synthetase binds to the AA and ATP
2. The ATP loses 2 P groups to become
AMP and binds with the AA
3. Then the right tRNA binds to the AA and
displaces the AMP
4. The enzyme then releases the “activated
AA”
20
RIBOSOMES:
SITES OF TRANSLATION
Ribosomes consist of 2 subunits, large and
small; they are composed of rRNA and proteins
They have 3 binding sites for tRNA:
E = about to exit
P = holds the AA chain
A = “on-deck” AA
The ribosomes itself catalyzes the peptide bond
between amino acids.
Like transcription,
translation can be
divided into 3
stages:
- initiation
- elongation
- termination
21
ENERGY SOURCE FOR TRANSLATION
GTP (guanosine triphosphate) → energy source for translation; this is very similar to
ATP and releases energy by breaking off phosphates
22
TRANSLATION - INITIATION
Steps:
1. Small ribosomal subunit binds to mRNA leader (5’ end)
2. Initiator tRNA (methionine) binds to “start” codon –
AUG
3. Next the large ribosomal subunit binds
4. All of these components (small unit, mRNA, tRNA, large
subunit) are brought together by initiation factors and
form the translation initiation complex
23
TRANSLATION - ELONGATION
3 step process for each AA:
1. Codon recognition
2. Peptide bond formation
3. Translocation
This process uses elongation
factors (proteins)
24
TRANSLATION - TERMINATION
When a stop codon (mRNA) gets to get the A-site and
instead of a tRNA binding, a release factor binds.
This adds a water molecule to the AA chain, and then
releases the chain from the ribosome.
After the chain is released, all the factors dissociate
from one another.
25
OVERVIEW –
PROTEIN
SYNTHESIS
26
POLYRIBOSOMES
OR
POLYSOMES
This is when many ribosomes trail along
the same mRNA molecule. They can
translate many proteins simultaneously
and therefore are much more efficient.
27
POST-TRANSLATIONAL MODIFICATIONS


During and after synthesis, a polypeptide spontaneously coils and
folds to its three-dimensional shape.
In addition, proteins may require post-translational modifications
before doing their particular job.
 These modifications may require additions such as sugars, lipids,
or phosphate groups to amino acids.
 In other cases, a polypeptide may be cleaved in two or more
pieces OR two or more polypeptides may join to form a protein
with quaternary structure.
28
SIGNAL PEPTIDES – DETERMINE WHETHER
RIBOSOME WILL BE ATTACHED OR FREE
All ribosomes start as free (in the cytosol); however, the polypeptide can cue the ribosome to
go attach to the ER and become bound. The signal peptide is a sequence of about 20 AA’s
near the front of the strand that tells the ribosome to go attach. This is the case for proteins/
enzymes that are going to be secreted from the cell. The signal recognition particle (SRP)
sees this signal peptide and brings the ribosome to the ER to attach.
29
MUTATIONS
Mutations are changes in the genetic material of a cell
(or virus). They are the ultimate source of new genes
(and genetic diversity!)
A point mutation (also called a substitution) is a change
in one base pair. It can have huge effects (sickle cell)
or no affect at all (silent mutation), depending on which
base is affected and where the AA is located in the
protein.
30
Missense = codes for a different AA
Nonsense = changes into a stop codon, so
it leads to a nonfunctional protein
Silent = changes the nucleotide but it codes
for the same AA
MISSENSE AND
NONSENSE MUTATIONS
31
FRAMESHIFT MUTATIONS
A frameshift mutation is when there is an insertion or deletion that causes the reading
frame to change. This means that all of the AA’s after the mutation will be wrong. It
has disastrous effects.
32
MUTATIONS CAN OCCUR DURING DNA
REPLICATION, DNA REPAIR, OR DNA
RECOMBINATION
Errors during DNA replication or recombination
can lead to nucleotide-pair substitutions,
insertions, or deletions.
 Mutagens are chemical or physical agents that
interact with DNA to cause mutations.
 Physical agents include high-energy radiation
like X-rays and ultraviolet light.
 Chemical mutagens cause mutations in
different ways.

33