Transcript From Gene to Protein

From Gene to Protein
DNA
RNA
Protein
What Is the Evidence that Genes Code for Proteins?
The molecular basis of phenotypes was
known before it was known that DNA is
the genetic material
Studies of many different organisms
showed that major phenotypic
differences were due to specific
proteins
Model organisms - easy to grow or
observe; show the phenomenon to be
studied
Examples: pea plants, Drosophila, E. coli,
common bread mold Neurospora crassa
Wild-type strains have enzymes to catalyze
all reactions needed to make cell
constituents - prototrophs
Beadle and Tatum used X-rays as mutagens.
Mutants were auxotrophs - needed
additional nutrients to grow
For each auxotrophic strain, they found a
single compound that would support
growth of that strain
Suggested the one-gene, one-enzyme
hypothesis
Beadle and Tatum found several different
arg mutant strains—had to be supplied
with arginine
arg mutants could have mutations in the
same gene; or in different genes that
governed steps of a biosynthetic
pathway
arg mutants were grown in the presence
of compounds suspected to be
intermediates in the biosynthetic
pathway for arginine
This confirmed that each mutant was
missing a single enzyme in the
pathway
The gene-enzyme relationship has been
revised to the one-gene, one-polypeptide
relationship
Example: In hemoglobin, each polypeptide
chain is specified by a separate gene
Other genes code for RNA that is not
translated to polypeptides; some genes are
involved in controlling other genes
Expression of a Gene to Form a
Polypeptide
• Transcription - copies information from
gene to a sequence of RNA
• Translation - converts RNA sequence to
amino acid sequence
RNA, ribonucleic acid differs from DNA:
• Usually one strand
• The sugar is ribose
• Contains uracil (U) instead of thymine (T)
RNA can pair with a single strand of
DNA, except that adenine pairs with
uracil instead of thymine
Single-strand RNA can fold into complex
shapes by internal base pairing
DNA
RNA
Protein
The central dogma of molecular biology information flows in one direction when genes
are expressed
Messenger RNA (mRNA) forms as a
complementary copy of DNA and
carries information to the cytoplasm
This process is transcription
Within each gene, only one strand of
DNA is transcribed - the template
strand
Transcription produces mRNA; the same
process is used to produce tRNA and
rRNA
The genetic code - specifies which amino
acids will be used to build a protein
Codon - a sequence of three bases.
• Each codon specifies a particular amino
acid
Start codon – AUG - initiation signal for
translation
Stop codons - stops translation and
polypeptide is released
For most amino acids, there is more than
one codon; the genetic code is
redundant
• each codon specifies only one amino
acid
The genetic code is nearly universal - the
codons that specify amino acids are the
same in all organisms
Exceptions: within mitochondria and
chloroplasts, and in one group of
protists
RNA polymerases catalyze synthesis of
RNA
RNA polymerases are processive - a
single enzyme-template binding results
in polymerization of hundreds of RNA
bases
Transcription occurs in three phases
1. Initiation
2. Elongation
3. Termination
Initiation
Requires a promoter - a special sequence
of DNA
RNA polymerase binds to the promoter
(commonly produces a TATA Box in
eukaryotes)
Promoter tells RNA polymerase where
to start, which direction to go in, and
which strand of DNA to transcribe
Elongation
RNA polymerase unwinds DNA about 10
base pairs at a time; reads template in
3′ to 5′ direction
The RNA transcript is antiparallel to the
DNA template strand
RNA polymerases do not proofread and
correct mistakes
Termination
Specified by a specific DNA base sequence
Eukaryotes - first product is a pre-mRNA
that is longer than the final mRNA and
must undergo processing
Transcription Animation
mRNA modification
1. 5’ cap - modified guanine; protection; recognition
site for ribosomes
2. 3’ tail - poly(A) tail (adenine); protection;
recognition; transport
3. RNA splicing - exons (expressed sequences) kept
introns (intervening sequences) are spliced out
forming a spliceosome
RNA Splicing
• Introns - intervening sequence
Non coding
• Exon - translates to Amino Acids
sequence
• Spliceosome – join together 2
exons that flank the intron
Ribozymes – RNA molecules that
function as enzymes
RNA Splicing
Animation
Evolutionary Importance??
Alternative RNA Splicing
• Gene gives rise to different proteins
depending on which segments are
exons during RNA processing
• Potentially new proteins with new
functions
Increase chance of crossing over between
genes
• increase genetic recombination
Transfer RNA (tRNA) - an adapter
molecule that can bind amino acids,
and recognize a nucleotide sequence
In the process of translation - tRNA
molecules carrying amino acids line up
on mRNA in proper sequence for the
polypeptide chain
Functions of tRNA:
• Carries an amino acid
• Associates with mRNA molecules
• Interacts with ribosomes
The conformation (3-D shape) of tRNA
results from base pairing (H bonds)
within the molecule
3′ end is the amino acid attachment site
- binds covalently. Always CCA.
Anticodon - site of base pairing with
mRNA. Unique for each type of
tRNA.
Example:
DNA codon for arginine: 3′-GCC-5′
Complementary mRNA: 3′-CGG-5′
Anticodon on the tRNA: 3′-GCC-5′ This
tRNA is charged with arginine
Ribosome - holds mRNA and tRNA in the
correct positions to allow assembly of
polypeptide chain
Ribosomes are not specific, they can
make any type of protein
Ribosomes have two subunits, large and
small
How Is RNA Translated into Proteins?
Large subunit has three tRNA binding
sites:
1. A site binds with anticodon of charged
tRNA (carrying an amino acid)
2. P site is where tRNA adds its amino
acid to the growing chain
3. E site is where tRNA sits before being
released
Hydrogen bonds form between the
anticodon of tRNA and the codon of
mRNA
Small subunit rRNA validates the match if hydrogen bonds have not formed
between all three base pairs, it must
be an incorrect match, and the tRNA is
rejected
Translation also occurs in three steps
1. Initiation
2. Elongation
3. Termination
Initiation
An initiation complex forms - charged
tRNA and small ribosomal subunit, both
bound to mRNA
rRNA binds to recognition site on mRNA
“upstream” from the start codon
Start codon is AUG - first amino acid is
always methionine (may be removed
after translation)
The large subunit joins the complex, the
charged tRNA is now in the P site of
the large subunit
Elongation
The second charged tRNA enters the A
site
Large subunit catalyzes two reactions:
1. Breaks bond between tRNA in P site
and its amino acid
2. Peptide bond forms between that
amino acid and the amino acid on
tRNA in the A site
When the first tRNA has released its
methionine, it moves to the E site and
dissociates from the ribosome - can
then become charged again
Elongation occurs as the steps are
repeated, assisted by proteins called
elongation factors
Termination
translation ends when a stop codon
enters the A site
Stop codon binds a protein release factor
- allows hydrolysis of bond between
polypeptide chain and tRNA on the P
site
Protein Synthesis Animation
Several ribosomes can work together to
translate the same mRNA, producing
multiple copies of the polypeptide
A strand of mRNA with associated
ribosomes is called a polyribosome or
polysome
What Are Mutations?
Somatic mutations occur in somatic
(body) cells. Mutation is passed to
daughter cells, but not to sexually
produced offspring
Germ line mutations occur in cells that
produce gametes. Can be passed to
next generation
All mutations are alterations of the
nucleotide sequence
Point mutations - change in a single base
pair
• loss, gain, or substitution of a base
Chromosomal mutations - change in
segments of DNA
• loss, duplication, or rearrangement
Point mutations can result from
replication and proofreading errors, or
from environmental mutagens
Silent mutations have no effect on the
protein because of the redundancy of
the genetic code
Silent mutations result in genetic
diversity not expressed as phenotype
differences
Missense mutations - base substitution
results in amino acid substitution
Nonsense mutations - base substitution
results in a stop codon
Frame-shift mutations - single bases
inserted or deleted
• usually leads to nonfunctional
proteins
Induced mutation - due to an outside
agent, a mutagen
Chemicals can alter bases (e.g., nitrous
acid can cause deamination)
Some chemicals add other groups to
bases (e.g., benzpyrene adds a group
to guanine and prevents base pairing).
DNA polymerase will then add any
base there
Ionizing radiation such as X-rays create
free radicals
• highly reactive
• can change bases, break sugar
phosphate bonds
UV radiation is absorbed by thymine,
causing it to form covalent bonds with
adjacent nucleotides
• disrupts DNA replication
Mutation provides the raw material for
evolution in the form of genetic
diversity
Mutations can harm the organism, or be
neutral
Occasionally, a mutation can improve an
organism’s adaptation to its
environment, or become favorable as
conditions change