Transcript GENE to PROTEIN

GENE to PROTEIN
• Garrod (1909) hypothesized that the symptoms
of an inherited disease reflect a person’s
inability to make a particular enzyme.
• The breakthrough in demonstrating the
relationship between genes and enzymes when
Beadle and Tatum began to search for mutants
of bread mold. They discovered that mutants
differ from wild type in their nutritional needs.
• Nutritional mutants are called auxotrophs.
• Beadle and Tatum were able to demonstrate the
relationship between genes and enzymes by
studying mutant forms of bread mold.
Each gene dictates the production of one enzyme.
• Many genes are made from two or more
polypeptide chains – therefore it is really one
gene – one polypeptide.
• Genes are typically hundreds or thousands of
nucleotides long. The nucleic acids and
proteins are written in two different languages.
DNA must be converted to protein language.
• This is accomplished through the processes of
• Transcription
• Translation
Prokaryotes vs Eukaroytes
• Prokaryotes lack nuclei and DNA is not
segregated from ribosomes and
transcription and translation occur in rapid
succession.
• Eukaryotes have a nuclear envelop envelop
and that segregates transcription in the
nucleus from translation that occurs in the
cytoplasm.
• The flow of information from a gene to a
protein is based a a triplet code.
• These three nucleotide “words” are codons.
Cracking the Genetic Code
• Nirenberg – 1961 NIH
• First codon decipher was UUU
• There are 64 codons
• A codon codes for only 1 amino acid
The genetic code must have evolved very early in
the history of life because it is nearly universal
among living organisms.
Transcription
• Transcription of messenger RNA from
template DNA is catalyzed by RNA
polymerase, which
• Separate the two strands of DNA and link RNA
nucleotides as they base-pair along the template
• Add nucleotides to the 3’ end; thus mNRA
grows in the 5’
3’ direction
• Transcription occurs in three stages
• Polymerase binding and initiation
• Elongation
• Termination
• In eukaryotes, RNA polymerase cannot recognize the
promoter ( about 100 nucleotides long) without
transcription factors.
• RNA polymerase II cannot recognize the promotor site
without binding to the TATA box ( a short sequence of
nucleotides rich in A and T that is about 25 nucleotides
upstream from the initiation site.
RNA SPLICING
• Introns – noncoding segments that lie
between the coding segments
• Exons -- coding segments that are
expressed
• Roberts and Sharp – independently
found evidence for “split gene” and both
received a Nobel Prize
• RNA polymerase transcribes all the
sequences and this is known as pre
mRNA. This pre mRNA never leaves the
nucleus.
• The introns are removed and the
“abridged” version of mRNA moves to
the cytoplasm as the primary
transcription.
• There are signals for the RNA splicing at
the ends of the introns. Particles called
small nuclear ribonucleoproteins
(snRNPs – “snurps”) begin the process.
• Several snRNA molecules join to form a
spliceosome ( about the size of a ribosome).
These cut the introns and connect the exons.
Ribozymes
• RNA molecules that function as enzymes
• Serve as a catalyst
• Makes the following statement obsolete:
“All biological catalyst are proteins.”
What is the function of Introns
and Splicing?
• May control gene activity and may
regulate passage of mRNA to cytoplasm
• May have a role in the evolution of new
proteins
• Increases the probability of crossing-over
• Split genes have a higher frequency of
recombination.
Translation
RNA directed synthesis of a polypeptide
• tRNA serves as the interpreter
• The role of tRNA is to transfer
amino acids from the “pool” in the
cytoplasm to a ribosome.
• The ribosome adds each amino acid
to the growning end of a polypeptide
chain ( 3’)
Transfer RNA
• Molecules of tRNA are not all identical
• Each type of tRNA links a mRNA codon
with a specific amino acid.
• One end has an attachment site for amino
acid and the anticodon is on the other end.
• May be used repeatedly
• Consists of a single RNA strand about 80
nucleotides long
• Looks like a clover leaf
• Loop protruding from L
end is the anticodon and
this binds to the mRNA
• There are 45 tRNA
molecules. ( Should be
61 if there was a tRNA
for each codon)
• Some tRNA can
recognize 2 or more
codons.
• WOBBLE – Allows for the
relaxation of the base-pairing rules
• U can pair with A or G in the third
position
• The most versatile tRNA has the
inosin (I). The I ( in the wobble
position) can form hydrogen bonds
with U, C or A
Steps in Translation
1. Correct match between tRNA
and a amino acid. The amino
acid is bound to the tRNA by
aminocyl tRAN synthetase.
There are 20 of these – one for
each amino acid
2. The codon-anticodon bonding.
Ribosomes
• 2 subunits
• Large subunit
• Small subunit
• 60% of weight of
ribosome is rRNA
• 3 Binding sites for tRNA
• P – holds the tRNA with
the growing polypetide
chain
• A – hold the next amino
acid to be added
• E – exit site
A single ribosome can make an average sized
polypeptide in less than one minute.
Usually a single mRNA is used to make many
copies of a polypeptide simultaneously
because several ribosomes work on
translating at the same time.
Polyribosome – string of ribosomes
.
Mutations
Change in the genetic makeup
• Point mutation – change in one or a few
base-pairs in a single gene
• If occurs in a gamete then the change is
passed to the future.
• If it has an adverse effect then it is known as
a genetic disorder or hereditary disease
Types of Point Mutations
•
Substitution
1. Misssense – still codes for an amino acid
2. Nonsense – does not code for an amino acid but
changes to a code for STOP. This makes the chain
too short and usually leads to nonfunctioning
proteins.
•Insertion or Deletion
1.Frameshift – not in a multiple of three
• Spontaneous mutation are the result of
errors in the DNA replication or repair.
• Mutagens are chemical or physical agents
that cause DNA to change.
• AMES test – is a test for mutagenic
activity of different chemicals.