Download 6 Protein_Synthesis - bloodhounds Incorporated

PHYSIOLOGY
Signal Transduction and Protein
Synthesis
DNA

DNA
– Deoxyribonucleic Acid
– Twisted ladder or double helix
– Nucleotides
» Composed of alternating sugar (Deoxyribose) and
phosphate molecules and
» Nitrogen bases


Purines = adenine and guanine
Pyrimidines = thymine cytosine
DNA

Purines bond with Pyrimidines
– Complementary base pairs
» Adenine with Thymine
» Guanine with Cytosine
DNA

Purines bond with Pyrimidines
– Complementary base pairs
» Adenine with Thymine
» Guanine with Cytosine

Nucleoside
– Sugar bonding with a base

Nucleotide
– Adding a phosphate to a nucleoside

Phosphates attach to the 5’ carbon of the sugar
Orientation of DNA
The carbon atoms on the sugar ring are
numbered for reference. The 5’ and 3’
hydroxyl groups (highlighted on the left)
are used to attach phosphate groups.

The directionality of a DNA strand is due to the
orientation of the phosphate-sugar backbone.
DNA is a double helix
P
T
A
P
G
5’
3’
P
A sugar and phosphate
“backbone” connects
nucleotides in a chain.
C
DNA has directionality.
PP
P
C
G
A
P
P
Two nucleotide chains
together wind into a helix.
T
P
P
G
Hydrogen bonds between
paired bases hold the two
DNA strands together.
C
P
P
3’
C
G
P
DNA strands are antiparallel.
5’
DNA

A chromosome
– 23 pair = diploid
– 23 = haploid; sex cells
– Duplicating DNA structure tightly packed
around histone proteins to form a nucleosome.
DNA

A gene
– A series of bases that occupy a specific location (locus) on a
chromosome
– The code of a single protein or polypeptide

Genetic Alphabet
– Triplet = Three nucleotides on DNA with their corresponding base
pairs making up the code of a single amino acid
– Codon = Three successive nucleotides on RNA with their
corresponding base pairs making up the code of a single amino
acid
– 20 amino acids
– A series of amino acids makes up a protein
DNA

Consists of 3 billion base pairs
– Codes for about 50 to 100,000 genes
– Genes may exist in alternate forms = alleles
» One allele from mom and one allele from dad
– Nucleotide changes or mutations may occur in a gene
» Sickle cell anemia
– In a healthy population, a gene may exist in multiple
alleles
– Genetic Polymorphism = Multiple different forms at a
gene locus in a population
» Basis for DNA typing using MHC
Terminology
– Allele
» An alternate form of a gene
– Locus
» Location of a gene on a chromosome
– Gene
» Genetic code or “blueprint” for the cell to build one
particular protein

http://www.youtube.com/watch?v=983lhh2
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http://www.youtube.com/user/ndsuvirtualce
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 http://www.youtube.com/watch?v=YjWuVr
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
http://www.youtube.com/watch?v=FVuAw
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http://www.youtube.com/watch?v=5bLEDd
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http://www.youtube.com/watch?v=NJxobgk
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Two types of nucleic acids
DNA
RNA
Usually
Has
single-stranded
uracil as a base
Ribose
as the sugar
Carries
protein-encoding
information
Can
be catalytic
Usually
Has
double-stranded
thymine as a base
Deoxyribose
Carries
as the sugar
RNA-encoding
information
Not
catalytic
Protein Synthesis
Proteins are necessary for cell functions
Protein synthesis is under nuclear direction
 DNA specifies Proteins
?
DNA
mRNA
?
Protein
Redundancy of Genetic Code (p 115)
A combination of three bases forms
a codon
1 start codon
3 stop codon
60 other codons for 19 aa
RNA

Definitions
– Exon
» Amino acid specifying informational sequences in
the genes of higher organisms
– Intron
» Noncoding segments or portions of DNA that ranges
from 60 to 100,000 nucleotides long
Transcription
DNA is transcribed into
complementary mRNA
by
RNA Polymerase
+ nucleotides
+ Mg2+
+ ATP
Gene = elementary
unit of inheritance
Compare to Fig. 4-33

http://vcell.ndsu.edu/animations/
Transcription
First steps in protein synthesis that occurs
completely within the nucleus
 DNA is used as a template to create a small
single strand of mRNA that can leave
through the nuclear pore.
 The enzyme RNA polymerase plus
magnesium or manganese ions along with
ATP are needed in this process.

Transcription
DNA is used as a template for creation of RNA using
the enzyme RNA polymerase.
DNA
5
’
G T C A T T C G G
3’
3’
C A G T A A G C C
5’
Transcription
The new RNA molecule is formed by incorporating
nucleotides that are complementary to the template strand.
DNA coding strand
5
’
DNA
G T C A T T C G G
3’
3’
G U C A U U C G G
3’
C A G T A A G C C
5’
DNA template strand
5
’
RNA
Transcription

Promoter
– Sequence on DNA where the RNA
polymerase attaches to begin
transcription
– A region at the beginning of a gene that must be
activated before transcription can begin.
– This region is not transcribed into mRNA
Transcription

Transcription Factors
– Binds to DNA and activates the promoter
» Tells the RNA polymerase where to bind to the DNA
» RNA polymerase moves along the DNA molecule and
“unwinds” the double strand by breaking hydrogen bonds
between base pairs
– Sense strand
» Guides RNA polymerase in RNA synthesis
– Antisense strand
» Sits idly by and is not transcribed
Transcription

Each base of the DNA sense strand pairs
with a complementary mRNA base
– AGTAC on DNA
– UCAUG on mRNA
Uracil is substituted for Thymine
 Ribose sugar is used as the backbone of
mRNA instead of Deoxyribose sugar

Initiation of transcription
Transcription begins at the 5’ end of the gene in a
region called the promoter.
The promoter recruits TATA protein,
a DNA binding protein, which in turn recruits
other proteins.
TATA binding protein
Promoter
DNA
GG
Transcription factor
Gene sequence
to be transcribed
TATA CCC
TATA box
Transcription begins
mRNA processing

Alternative splicing occurs
– Enzymes clip segments out of the middle or off
the ends of mRNA strands
» Introns
– mRNA segments are spliced back together by
the spliceozyme enzyme
» Exons

The processes mRNA leaves through the
nuclear pore and attaches to a ribosome
mRNA
Contains the coded information for the
amino acid sequence of a protein
 3 main parts:

– 5' leader sequence - important for the start of
protein synthesis.
– Coding Sequence - the sequence that codes for
the amino acid.
– 3’ trailer sequence - poly A tail.
Messenger RNA undergoes three (or
four) post-transcriptional
modifications
1. Capping of 5’ end
2. Additional of poly A tail to 3’ end
3. Removal of introns
4. Editing of RNA (rarely)
EUKARYOTES ONLY!!!!!!!!!!!!!!!!
5’ capping.
Involves the addition of a guanine (usually 7-methylguanosine) to the terminal 5’ nucleotide.
 The enzyme that completes this process is called a
capping enzyme.
 The 5’ cap is required for the ribosome to bind to the
mRNA as the initial step of translation.
Addition of a 3’poly A tail.





This poly(A) tail is usually about 50 - 250 bps of adenine in
length.
There is no DNA template for this tail?
Poly A tails are found on most mRNA molecules but not all
(ex. histones mRNA have no poly A tail).
In general, a eukaryotic mRNA molecule is longer than the
required transcript. The enzyme RNA endonuclease cleaves
the molecule at the poly(A) addition site to generate a 3’ OH
end.
The poly A tail is important for determining the stability of the
mRNA molecule so the mRNA doesn’t degrade.
Translation

Translation begins when mRNA binds to a
ribosome in the cytoplasm of the cell.
Translation
mRNA is translated into string of aa (= polypeptide)
2 important components ??
mRNA + ribosomes + tRNA meet in cytoplasm
Anticodon pairs with mRNA
codon  aa determined
Amino acids are linked via
peptide bond.
The Genetic Code

The code has start and stop signals.
AUG (methionine) is the common start codon
Methionine can also be used WITHIN a polypeptide
GUG may also be used as a start codon.

There are three stop codons.
UAG
UAA
UGA
All three are chain termination codons.
Ribosomal RNA
Large and small subunits
 Binding sites

– One for mRNA
– Three for tRNA
» P site = Peptidyl-tRNA site
» A site = Aminoacyl-tRNA site
» E site = Exit site
Transfer RNA

The correct amino acid is added to the
growing polypeptide only if:
– 1 - The appropriate amino acid is added to the
tRNA by aminoacyl-tRNA synthetases.
– 2 – Complementary binding occurs between the
codon of the mRNA and the anticodon of the
tRNA.
Translation (An Overview)





Translation is defined as protein synthesis.
Occurs on ribosomes, where the genetic information
is translated from the mRNA to a protein.
mRNA is translated in the 5’ to 3’ direction.
Amino acids are brought to the ribosome bound to a
specific tRNA molecule.
The mRNA and tRNA are responsible for the correct
recognition of each amino acid in the growing
polypeptide
Initiation

A small ribosomal subunit binds to both
mRNA at the 5’ cap along with a specific
initiator tRNA
– The initiator tRNA carries methionine
tRNA’s anticodon binds with the codon on
mRNA
 The large ribosomal subunit attaches to form
the translation initiation complex.
 The initiation complex is held together by
proteins called initiation factors

Initiation
The tRNA sits in the P site of the ribosome
 The A site is vacant
 The methionine is at the N-terminus of the
growing protein
 The carboxyl end is called the C-terminus
 All proteins grow from the N to the C-terminus

Elongation

Binding of the aminoacyl-tRNA to the ribosome

formation of a peptide bond

The movement (translocation) of the ribosome along
the mRNA, one codon at a time.
Elongation

Three step cycle
– The ribosome will move 5’ to 3’ on the mRNA

Step one
– The anticodon of an incoming aminoacyltRNA
base-pairs with the complementary mRNA codon
in the A site
– GTP hydrolysis occurs
Elongation

Step two
– The large ribosomal subunit catalyzes the formation of a
peptide bond
– Hydrogen bonds break between the t-RNA in the P site and
between the codon and anti-codon

Step three – translocation
– The ribosomes moves along the mRNA one codon
– The tRNA that was in the A site is now in the P site
– The tRNA in the P site exits through a tunnel in the rRNA
called the E site
– The next tRNA enters in the empty A site
Termination
Termination is usually signaled by one of the
three stop codons UAG, UAA or UGA.
 There are a number of “helper” proteins
involved (e.g. termination factors and release
factors).
 GTP is necessary to break the complex apart

Translation initiation
Leader
sequence
Small ribosomal subunit
5’
3’
mRNA
mRNA
U U C G U C A U G G G A U G U A A G C G A A
U A C
Assembling to
begin translation
Initiator tRNA
Met
Translation Elongation
Ribosome
5’
3’
mRNA
A U G G G A U G U A A G C G A
U A C C C U
tRNA
P
A
Amino acid
Met
Gly
Large ribosomal subunit
Translation Elongation
5’
3’
mRNA
A U G G G A U G U A A G C G A
U A C C C U
A
P
Met
Gly
Translation Elongation
5’
3’
mRNA
A U G G G A U G U A A G C G A
C C U A C A
P
Gly
Cys
A
Translation Elongation
5’
3’
mRNA
A U G G G A U G U A A G C G A
P
C C U A C A
Gly
Cys
A
Translation Elongation
5’
3’
mRNA
A U G G G A U G U A A G C G A
A C A U U C
P
Cys
Lengthening
polypeptide
(amino acid chain)
Lys
A
Translation Elongation
5’
3’
mRNA
A U G G G A U G U A A G C G A
A C A U U C
P
Cys
Lys
A
Translation Elongation
5’
3’
mRNA
A U G G G A U G U A A G C G A
A C A U U C
P
Cys
Lys
A
Translation Elongation
Stop codon
5’
mRNA
A U G G G A U G U A A G C G A U A A
U U C
A
P
Lys
Release
factor
Translation Termination
Stop codon
Ribosome reaches stop codon
5’
mRNA
A U G G G A U G U A A G C G A U A A
P
Release
factor
A
Translation Termination
Once stop codon is reached,
elements disassemble.
Release
factor
P
A
Translation Modifications
Protein folding
 Glycosylation

– Addition of glycogen to the protein by the
Golgi Apparatus
– Create a glycoprotein

Vesiculation
– Protein is surrounded by a vesicle

Exocytosis will then occur
Post – Translational protein modifications:
Folding, cleavage, additions  glyco- , lipo- proteins
Protein Sorting

No signal sequence  protein stays in cell

Signal sequence  protein destined for translocation into
organelles or
for export
For “export proteins”: Signal sequence leads
growing polypeptide chain across ER membrane
into ER lumen
Modifications in ER
Transition vesicles to
Golgi apparatus for further
modifications
Transport vesicles to cell
membrane
Signal Transduction
1st The signal molecule is a ligand that binds to a
receptor. The ligand is also known as the first
messenger because it brings information to its
target cell
2nd Ligand-receptor binding activates the receptor
3rd The receptor in turn activates one or more
intracellular signal molecules
4th the last signal molecule in the pathway initiates
synthesis of target proteins or modifies existing
target proteins to create a response
Receptor Proteins

Lipophilic signal molecules
– Can diffuse through the phospholipid bilayer
and bind to cytosolic receptors or nuclear
receptors
– Steroids are lipophilic

Lipophobic signal molecules
– Unable to diffuse through the phospholipid
bilayer of the cell
– Bind to receptor proteins on the cell membrane
Receptor-Enzymes


Transmembrane receptor binds with a ligand on
the extracellular surface of the cell
Intracellularly an enzyme is bound to the receptor
protein
– The enzyme is typically a protein kinase (ie. tyrosin
kinase) or guanylyl cyclase
– Guanylyl cyclase converts GTP to cyclic GMP (cGMP)
– Adenylyl cyclase converts ATP to cyclic AMP (cAMP)
Signal Transduction

The process by which an extracellular
signal molecule activates a membrane
receptor that in turn alters intracellular
molecules to create a response
Signal Amplification

Turns on signal molecules into multiple
second messenger molecules
Steps of Signal Transduction
An extracellular signal molecule binds to
and activates a protein or glycoprotein
membrane receptor
 The activated membrane receptor turns on
its associated proteins

– The proteins may activate protein kinases
– The proteins may create an intracellular second
messenger
Second Messenger

Second messenger molecules
– Alter the ion channels by opening or closing
them
– Increase intracellular calcium in order for the
calcium to bind to proteins and change their
function
– Change enzyme activity

Signal molecule binds to the G-protein linked
receptors
– The protein changes confirmation and activates the
intracellular G protein




The G protein moves horizontally in the
membrane to bind with adenylyl cyclase, an
amplifier enzyme
Adenylyl cyclase converts ATP to cyclic AMP
cAMP activates protein kinase A
Protein kinase A phosphorylates other proteins
– There is a cellular response
» Such as a protein binding to the promoter site on DNA to start
transcription
» Release of calcium to change enzyme activity
Specificity v Competition

Receptors have binding sites for ligands
– Different molecules may be able to bind to the
same receptor
» Ie. Epinephrine and its cousin Norepinephrine

These both bind to a class of receptors called Adrenergic
receptors
– Alpha and Beta receptors
» Alpha has a higher affinity for norepinephrine
» B2 receptors have a higher affinity for epinephrine
Agonists v Antagonists

When a ligand combines with a receptor
– Either the ligand turns the receptor on and
elicits a response or
– The ligand occupies the binding site and
prevents a response from happening
Agonist – turns receptors “on”
 Antagonist – turns receptors “off”

Which of the following
nucleotide bases in DNA can
form H-bonds with the base
adenine?
A.
Thymine
B.
Uracil
C.
Guanine
D.
Cytosine
E.
Both A and B
Which of the following nitrogen
bases are purines?
A. Uracil and Guanine
B. Adenine and Thymine
C. Guanine and Cytosine
D. Cytosine and Adenine
E. Adenine and Guanine
How many pair of chromosomes
are found in a diploid cell?
A.
B.
C.
D.
E.
8
16
23
46
0
DNA is formed by hydrogen
bonding two antiparallel strands.
A.
B.
True
False
In which direction is a DNA
strand read?
A.
B.
C.
D.
5’ to 3’
3’ to 5’
3’ to 3’
5’ to 5’
In DNA, guanine is bonded to
cytosine by
A.
B.
C.
D.
ionic bonding
coordinate covalent bonding
covalent bonding
hydrogen bonding
A gene can best be defined as:
A. Three base triplet that specifies a particular
amino acid
B. Non-coding segments of DNA up to 100,000
nucleotides long.
C. A segment of DNA that carries the
instructions for one polypeptide chain.
D. An RNA messenger that codes for a particular
polypeptide.
If the nucleotide or base sequence of the DNA
strand used as a template for messenger RNA
synthesis is ACGTT, then the sequence of
bases in the corresponding mRNA would be:
A.
B.
C.
D.
TGCAA
ACGTT
UGCAA
GUACC
In DNA, complementary base pairing occurs
between _______________
A.
B.
C.
D.
Cytosine and thymine
Adenine and guanine
Thymine and uracil
Guanine and cytosine
Messenger RNA
A.
B.
C.
D.
is composed of two nucleotide chains
similar to DNA
is a very stable molecule; that is, it is not
easily broken down
transfers genetic information from DNA
molecules to the ribosome
is synthesized on the ribosomes in the
cytoplasm
Transcription
A. Occurs on the surface of the ribosome
B. Is the final process in the assembly of a
protein
C. Occurs during the synthesis of any type of
RNA by use of a DNA template
D. Is catalyzed by DNA polymerase
___________ is an enzyme that breaks the
hydrogen bonds on DNA to begin the process
of transcription
A.
B.
C.
D.
E.
DNA polymerase
RNA polymerase
Lipase
Phenylketonurinase
Aldolase
The genetic code on mRNA for
methionine is
A.
B.
C.
D.
AUG
AUC
UAG
UAC
Where on DNA does cAMP
bind?
A.
B.
C.
D.
Initiator site
Promoter site
DNA binding site
TATA box