Download Figure 5.x3 James Watson and Francis Crick

FROM GENE TO
PROTEIN
Chapter 17
Experiments Leading to Understanding
the Link between Genes and Proteins

Nirenberg & Matthaei – determining the
language of the genetic code.
 http://bcs.whfreeman.com/thelifewire/content/
chp12/1202002.html

Beadle & Tatum – One Gene…One
Enzyme hypothesis.
 http://www.dnalc.org/view/16360-Animation-
16-One-gene-makes-one-protein-.html
What is a Gene?

Functional definition – a DNA sequence coding
for a specific polypeptide chain
TEXT PAGE 324 – LEARN THE OVERVIEW…IT
WILL HELP YOU MAKE MEANINGFUL
UNDERSTANDING OF PROTEIN SYNTHESIS!!!

HELPFUL WEBSITES:


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 http://highered.mcgraw-
hill.com/olc/dl/120077/micro06.swf

Google – “TRANSCRIPTION & TRANSLATION ANIMATIONS”
Overview of Protein Synthesis



Genes contain the blueprints for building
proteins in cells.
RNA is the bridge between DNA and its protein.
The “genetic code” is the sequence of bases in
DNA that will code for specific amino acids in a
growing polypeptide (protein).
 There
are 20 possible amino acids.
Types of RNA

RNA is a nitrogenous base that consists of a
ribose (5 carbon sugar).



In most cells, RNA molecules are involved in just
one job – protein synthesis!


In RNA, the base Uracil is substituted for Thymine in
DNA.
The RNA nucleic acid is single stranded.
The assembly of amino acids into proteins is
controlled by RNA
There are 3 main types of RNA:
1.
2.
3.
Messenger RNA (mRNA)
Ribosomal RNA (rRNA)
Transfer RNA (tRNA)
Protein Synthesis

2 stages: Transcription and Translation

Transcription occurs in the nucleus
 DNA  mRNA
 Making mRNA from a DNA template in the nucleus.

Translation occurs in the cytoplasm at a
ribosome
 mRNA  protein (using tRNA and rRNA)
 Translating instructions in mRNA into a growing polypeptide
protein chain at the ribosome.
Prokaryote vs Eukaryote
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

Protein Synthesis is
about the same in
both cases.
Here the two steps
of Transcription an
Translation occur in
a bacteria.
Prokaryote vs Eukaryote



In Eukaryotes one
more step occurs.
mRNA will be
processed before it
leaves the nucleus.
Segments of mRNA
that are not needed
will be removed.
RNA and Protein Synthesis

TYPES OF RNA



Messenger RNA (mRNA) – carries information from DNA in
the nucleus to the ribosomes where the proteins are
assembled. It is a partial copy of ONLY the information
needed for that specific job. It is read 3 bases at a time –
codon.
Ribosomal RNA (rRNA) – found in ribosomes and helps in
the attachment of mRNA and in the assembly of proteins.
Transfer RNA (tRNA) – transfers the needed amino acids
from the cytoplasm to the ribosome so the proteins dictated by
the mRNA can be assembled. (The three exposed bases are
complementary to the mRNA and are called the
anticodon).
Types of RNA
rRNA
Figure 5.28 DNA → RNA → protein: a diagrammatic overview of information
flow in a cell
Figure 17.3 The triplet code
For each gene, one DNA strand
functions as a template for
transcription – the synthesis of
a complementary mRNA
molecule.
Uracil takes the place of thymine
in RNA.
During translation, the mRNA is
read as a sequence of base
triplets (codons).
Each codon specifies an amino
acid to be added to the
growing polypeptide chain.
Genes are read in the 3’ to 5’
direction, so mRNA is made in
the 5’ to 3’ direction!
The Transfer of the Genetic Code
The amount of nucleotides that code for
an amino acid has to consist of THREE
bases.
 The three amino acid sequence on the
mRNA strand is termed a CODON.
 There are more than one codon for each
amino acid due to the 64 combinations
possible and only 20 amino acids.

Where and What is Happening?


The gene will
transcribe its self into a
mRNA strand each
triplet (codon) will code
for a specific amino
acid.
But, How do we know
what amino acid?
The Codon Chart


We can then see
what codon will
produce what
amino acid.
You will have to
do this.
 Ex.
AGG
 Ex. CCU
 Ex. CCT
 Point

A particular triplet of bases in the coding sequence of
DNA is AGT. The corresponding codon for the mRNA
transcribed is:






AGT
UCA
TCA
AGU
Either UCA or TCA, depending on wobble in the first base
Answer: UCA
 Point

A possible sequence of nucleotides in DNA that
would code for the polypeptide sequence pheleu-ile-val would be:
 5’
 3’
TTG-CTA’CAG’TAG 3’
AAC-GAC-GUC-AUA 5’
 5’ AUG-CTG-CAG-TAT 3’
 3’ AAA-AAT-ATA-ACA 5’
 3’ AAA-GAA-TAA-CAA 5’

Answer: 3’ AAA-GAA-TAA-CAA 5’
 Point

Which of the following is correct about a codon?
It…
 Consists of two nucleotides
 May code for the same amino acid as another
 Consists of discrete amino acid regions
 Catalyzes RNA synthesis
 Is the basic unit of the genetic

codon
code
Answer: may code for the same amino acid as
another codon.
Section 12-3
Transcription
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Adenine (DNA and RNA)
Cystosine (DNA and RNA)
Guanine(DNA and RNA)
Thymine (DNA only)
Uracil (RNA only)
RNA
polymerase
DNA
RNA
During transcription, RNA polymerase uses one strand of DNA as a template
to assemble nucleotides into a strand of RNA.
Figure 17.6 The stages of transcription: initiation, elongation, and termination (Layer 4)
Promoters and Terminators




Promoter: sequence in DNA where RNA
polymerase attaches and initiates transcription
Terminator: sequence that signals the end of
transcription
Transcription Unit: stretch of DNA that is being
copied into mRNA
RNA Transcript: stretch of mRNA created using
DNA as a template.
 Point

Where is the attachment site for RNA
polymerase?
 Structural
gene region
 Initiation region
 Promoter region
 Operator region
 Regulator region

Answer: promoter region
Figure 17.7 The initiation of transcription at a eukaryotic promoter
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wire/content/chp14/1402002.html
 Point

Which of the following is LEAST related to the
other items?
 Translation
 TATA
box
 Transcription
 Template strand
 RNA polymerase II

Answer: translation
RNA Processing
Enzymes in the Eukaryotic nucleus must
modify pre-mRNA before the genetic
message is dispatched to the cytoplasm.
 This process is called RNA PROCESSING:

 Both

ends of the primary transcript are altered.
Addition of 5’ cap and Poly (A) tail
 Certain
interior sections of the molecule are cut
out and then the remaining parts are spliced
together – called RNA SPLICING.
Figure 17.8 RNA processing; addition of the 5 cap and poly(A) tail
A modified guanosine
triphosphate added to
the 5’ end.
50 to 250 adenine
nucleotides added to the
3’ end created by
cleavage downstream of
the termination signal.
•Enzymes modify the two ends of a eukaryotic pre-mRNA molecule.
•The modified ends help protect the RNA from degradation, and the poly(A) tail may promote the
export of mRNA from the nucleus.
• When mRNA reaches the cytoplasm, the modified ends, in conjunction with certain cytoplasmic
proteins, facilitate ribosome attachment.
•The leader and trailer are not translated, nor is the poly(A) tail.
Figure 17.9 RNA processing: RNA splicing
The gene shown here and its pre-mRNA transcript have three regions, called
exons, that consist mostly of coding sequences; exons are separated by
noncoding regions, called introns.
During RNA processing, the introns are excised and the exons are spliced
together.
 Point

What are the coding segments of a stretch of
eukaryotic DNA called?
 Introns
 Exons
 Codons
 Replicons
 Transposons

Answer: exons
How is Pre-mRNA Splicing Done?

snRNP’s : small nuclear ribonucleoproteins
 these
look for special sequences at ends of introns to
know where to cut
 made up of snRNA (small nuclear RNA)

Several different snRNP’s join with additional
proteins to form the splicesome – this interacts
with the splice sites at intron ends, and cuts
them out – then joins the two exons together.

http://bcs.whfreeman.com/thelifewire/conte
nt/chp14/1402001.html
Figure 17.10 The roles of snRNPs and spliceosomes in mRNA splicing
1. Pre-mRNA
containing exons
and introns
combines with
small nuclear
ribonuceloprotei
ns (snRNPs)
and other
proteins to form
a molecular
complex call
spliceosome.
2. Within the
spliceosome,
snRNA basepairs with
nucleotides at
the ends of the
intron.
3. The RNA
transcript is cut
to release the
intron, and the
exons are
spliced together.
The
spliceosome
then comes
apart, releasing
mRNA, which
now contains
only exons.
Translation

Translation is the RNA-directed synthesis of a
polypeptide.
Generally: is the reading of the codons on the mRNA
strand and the sequencing of them into an amino acid
sequence – polypeptide.

The Players:




mRNA: already processed within the nucleus, will be the template
for the sequence of amino acids.
tRNA: transfers Amino acids from the Cytoplasm to the ribosome.
Ribosome: adds amino acids together from the tRNA and in the
sequence of the mRNA.
Figure 17.15 The anatomy of a functioning ribosome
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Structure:
It is made out of RNA and proteins.
The largest type of RNA is ribosomal
RNA (rRNA). It has two subunits
which will combine to form a ribosome
in the cystol.
Each ribosome has a binding site for
mRNA and three binding sites for tRNA
molecules.
The P site holds the tRNA carrying the
growing polypeptide chain.
The A site carries the tRNA with the
next amino acid.
Discharged tRNAs leave the ribosome
at the E site.
 Point

What are ribosomes composed of?
 Two
subunits, each consisting of rRNA only
 Two subunits, each consisting of several proteins only
 Both rRNA and protein
 mRNA, rRNA, and protein
 mRNA, tRNA, rRNA, and protein

Answer: both rRNA and protein
Figure 17.13a The structure of transfer RNA (tRNA)
tRNA is made in the nucleus.
Function:
Pick up designated amino
acids in the cystol.
Deposit the amino acid at
the ribosome. Return to
the cystol to pick up
another amino acid.
Structure:
It is made out of about 80
nucleotides.
It is folded in on its self in
a T shape.
One important look is
called the anti codon.
This base pairs with the
codon on the mRNA
strand.
tRNA


Amino Acids are
placed onto the tRNA
by the enzyme
aminoacyl tRNA
synthase.
There are 20 different
synthases for the 20
different types of
amino acids.
 Point

What is an anticodon a part of?
 DNA
 tRNA
 mRNA
A
ribosome
 An activating enzyme

Answer: tRNA
Translation

Translation can be divided into three steps:
 1.
Initiation

 2.
Elongation

 3.
Requires energy (GTP)
Requires energy (GTP)
Termination
Translation
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Nucleus
Messenger RNA
Messenger RNA is transcribed in the nucleus.
Phenylalanine
tRNA
The mRNA then enters the cytoplasm and
attaches to a ribosome. Translation begins at
AUG, the start codon. Each transfer RNA has
an anticodon whose bases are complementary
to a codon on the mRNA strand. The ribosome
positions the start codon to attract its
anticodon, which is part of the tRNA that binds
methionine. The ribosome also binds the next
codon and its anticodon.
Ribosome
Go to
Section:
mRNA
Transfer RNA
Methionine
mRNA
Lysine
Start codon
Translation Continued
Translation Initiation
• The initiation stage of translation brings together mRNA, a
tRNA bearing its first amino acid of the polypeptide, and the
two subunits of a ribosome.
• All three parts need to be in place for initiation.
– Initiator tRNA (Met), Small Ribosomal subunit and Large Ribosomal
subunit.
• Energy is needed to bind the tRNA to the P site (GTP).
Figure 17.17 The initiation of translation
A small ribosomal subunit binds to a
molecule of mRNA. An initiator tRNA,
with the anticodon UAC, base-pairs with
the start codon, AUG. This tRNA
carries the amino acid methionine (Met).
The arrival of a large ribosomal
subunit completes the initiation
complex. Proteins call initiation
factors are required to bring all the
translation components together. GTP
provides the energy for assembly. The
initiator tRNA is in the P site; the A site
is available to the tRNA bearing the
next amino acid.
Translation Elongation – Text 318

Translation elongation occurs in three steps:
1.
Codon Recognition


2.
Peptide Bond Formation


3.
The mRNA codon in the A site of the ribosome forms a H-bond with the
anticodon of an incoming molecule of tRNA carrying its appropriate
amino acid.
This requires energy. (GTP) (A site)
The ribosome catalyzes the formation of a peptide bond between the
new amino acid and the growing polypeptide.
Catalyzed by the ribosome.
Translocation




The tRNA in the P site and A site are now moved to the E site and P
site respectively.
The tRNA in the E site will detach and a new codon is open.
The ribosome shifts the mRNA by one codon “reading it”
This step requires energy. (GTP)
Figure 17.18 The elongation cycle of translation
Codon Recognition
An incoming tRNA binds to
the codon in the A site by a
H-bond.
Peptide Bond Formation
The ribosome catalyzes
the formation of a peptide
bond between the new
amino acid and the
growing polypeptide.
Translocation
The tRNA in the A site is
translocated to the P site,
taking the mRNA along
with it. Meanwhile the
tRNA in the P site moves to
the E site and is released
from the ribosome. The
ribosome shifts the mRNA
by one codon.
Figure 17.19 The termination of translation
When a ribosome
reaches a termination
codon on mRNA, the A
site of the ribosome
accepts a protein called
a release factor instead
of tRNA.
The release factor
hydrolyzes the bond
between the tRNA and
the P site and the last
amino acid of the
polypeptide chain. The
polypeptide in thus
freed from the
ribosome.
The two ribosomal
subunits and the
other components
of the assembly
dissociate.
 Point

What is the function of the ribosome in
polypeptide synthesis?

Answer:
 Holds
mRNA and tRNAs together
 Catalyzes the addition of amino acids from the tRNAs
to the growing polypeptide chain
 Moves tRNA and mRNA during the translocation
process
From Polypeptide to Functional Protein

During and after its synthesis, a polypeptide
chain begins to coil and fold spontaneously,
forming a functional protein of specific
conformation:
A
three-dimensional molecule with secondary
and tertiary structure.
A gene determines the primary structure.
 The primary structure in turn determines conformation.
 In many cases, a chaperone protein helps the
polypeptide fold correctly.

 Point

Which of the following is NOT directly involved in
the process of translation?
 Ligase
 tRNA
 rRNA
 mRNA
 Aminoacyl-tRNA

Answer: ligase
synthase
Review – PROTEIN SYNTHESIS

Transcription
 mRNA (in nucleus)
 In eukaryotes will have RNA processing (in nucleus).
 DNA

Translation
 mRNA

 Polypeptide (at ribosome in cytoplasm)
Coiling & Folding
 Three
dimensional (in cytoplasm as translation is
occurring)
 Chaperone proteins involved
Figure 17.4 The dictionary of the genetic code
Section 12-3
Go to
Section:
The Genetic Code
Polyribosomes

A single ribosome can make an averagesized polypeptide in less than a minute;
 Typically,
however, a single mRNA is used to
make many copies of a polypeptide
simultaneously, because a number of
ribosomes work on translating the message at
the same time.
 Multiple ribosomes may trail along the same
mRNA (in strings called polyribosomes).

These help a cell make many copies of a
polypeptide very quickly.
Mutations

Mutations are changes in the genetic
material of a cell.


Read text pages 322, 323, 325
Point Mutations are chemical changes in
just one base pair of a gene. There are two
types of point mutations:
 Base-Pair



Base-pair substitution
Missense mutation
Nonsense mutation
 Base-Pair

Substitutions:
Insertions & Deletions:
Frameshift mutation
Base-Pair Substitutions

Base pair substitution is the replacement of one
nucleotide and its partner in the complementary
DNA strand with another pair of nucleotides.
 Some
substitutions have no effect on the genetic code
– it may transform one codon into another that is
translated into the same amino acid.

CCG mutated to CCA – mRNA codon GGC will become GGU
– both code for glycine.
 Some
may switch the amino acid but have little effect
on the protein (if amino acid properties are similar).
 Some may cause a detectable change in a protein:


Missense mutations (wrong amino acid coded for & therefore
dysfunctional protein produced)
Nonsense mutations (amino acid codon changed to a stop
codon – no protein produced)
Base-Pair Insertions & Deletions

Insertions and deletions are additions or losses
of nucleotide pairs in a gene.
 These
mutations have disastrous effect on the resulting
protein b/c these change the entire triplet code being
read – ALL THE WAY DOWN THE mRNA LINE!
 This occurs when the reading frame (triplet grouping) is
altered.



Called a frameshift mutation
All nucleotides downstream of the mutation will be improperly
grouped into codons
Unless the frameshift is near the end of a gene, it will produce
a protein that is almost certain to be nonfunctional!
Gene Mutations:
Substitution, Insertion, and Deletion
Deletion
Substitution
Go to
Section:
Insertion
Gene Mutations that only affect ONE point of the code -- often
called Point Mutations
Figure 17.24 Categories and consequences of point mutations: Base-pair insertion
or deletion
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mations/ch13a08.htm
Chromosomal Mutations – affects LARGE portions of the code
(entire genes or entire chromosome).
Deletion
Duplication
Inversion
Translocation
Go to
Section:
 Point

A frameshift mutation could result from:
A
base insertion only
 A base deletion only
 A base substitution only
 Deletion of 3 consecutive bases
 Either an insertion or a deletion of a base

Answer: either an insertion or a deletion of a
base
So, what is a gene?

Functional definition – a DNA sequence coding
for a specific polypeptide chain
TEXT PAGE 324 – LEARN THE OVERVIEW…IT
WILL HELP YOU MAKE MEANINGFUL
UNDERSTANDING OF PROTEIN SYNTHESIS!!!

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 Point

What is the relationship among DNA, a gene, a
chromosome, proteins and phenotypes?

Answer: A chromosome contains hundreds of
genes, which are composed of DNA. The DNA
is used as a template for building proteins.
Expression of proteins generates certain
phenotypes in individuals.
The Essay – IT’S A DOOZY!

A portion of a specific DNA molecule consists of the
following sequence of nucleotide triplets:
TAC

GAA
CTT
CGG
TCC
This DNA sequence codes for the following short
polypeptide:
methionine - leucine - glutamic acid - proline - arginine
a)
b)
c)
d)
Describe the steps in the synthesis of this polypeptide.
What would be the effect of a deletion or an addition in one of the DNA
nucleotides?
What would be the effect of a substitution in one of the nucleotides?
Cells regulate both protein synthesis and protein activity. Discuss TWO
specific mechanisms of protein regulation in eukaryotic cells.