Molecular Biology of the Gene
Genes are biological blueprints. They give us our attributes & traits. Every
nucleus, in every cell carries the genetic blueprint for dictating who we are. Every
cell has all the information needed to make a complete you. Genes are located on
chromosomes. Humans have 46 chromosomes each containing thousands of genes.
We share gene sequences with all living organisms. 98% of our sequences match
chimpanzees and 99.9% match all other humans. Differences exist at particular
sites which cause each of us to be unique. Differences maybe as small as one base
substitution in one gene. Genes are made of DNA, a coiled, double strand of
deoxyribonucleic acid. This macromolecule is made of 4 different nucleotides that
are paired in a precise manner. The order of nucleotides is the genetic code. Each 3
combinations of nucleotides = one amino acid. Amino acids are the monomers of
proteins. DNA therefore gives instructions to make proteins in the body. The
smallest chromosome, the Y chromosome has 50 million nucleotides and the
largest has 250 million.
DNA is a nucleic acids. Nucleic acids are large macromolecule composed of
smaller subunits called nucleotides. Each nucleotide contains a 5 carbon sugardeoxyribose, a nitrogenous base and 1-3 PO4 groups. DNA contains 4 different
nucleotides each with a different nitrogenous base. These bases are found in 2
major groups: purines, single ring structures including adenine (A), guanine (G)
and pyrimidines, double ring structures which include thymine (T) and cytosine
(C). Bases are linked via dehydration synthesis by phosphodiester bonds in which
the phosphate of one nucleotide bonds covalently to the sugar of the next forming a
sugar-PO4 backbone. The nitrogenous bases are arranged as appendages along the
backbone.
The 3-D structure of DNA was determined by Watson and Crick in 1953 for which
they won the Nobel Prize in 1962. They discovered that DNA is a double stranded
helix. It is composed of two strands wrapped around each other in a helical
formation. At the core are the bases of one DNA strand bonded to the bases in the
other strand. If you think of the DNA molecule as a ladder the sugar-phosphate
backbone would be the sides of the ladder and the paired bases would be the rungs.
Base pairing is very specific: A-T only and G-C only. In a DNA molecule the
amount of A = amount of T. One strand is complementary to the other.
Replication
Cells divide and reproduce themselves daily giving rise to 2 daughter cells with the
same genetic makeup. Before a cell can divide, DNA must duplicate and make
copies of its self. This is called replication and uses a template mechanism. From
the structure of DNA and because the pairing of bases is specific, if you know the
sequence of one strand you can determine the sequence of bases in the other strand
by applying the rules of base pairing. Cells apply the same rules to copy genes
before cell division. During replication the strands must separate. To facilitate
separation a portion of the double helix is unwound by the enzyme helicase which
breaks H bonds between base pairs. This unwinding takes place in a replication
bubble and a new strand of DNA is formed in both directions on both strands of
DNA in the bubble. There are many areas where replication can begin which
speeds the replication process.
Each strand of DNA can be used as a template to make a new, complementary
strand using a supply of nucleotides. Replication proceeds in both directions.
Each strand of DNA has a 3’ end and a 5’ end. At one end carbon 3 of the sugar is
attached to a –OH group and at the other end carbon 5 is attached to a phosphate
group. DNA polymerase the enzyme that binds single nucleotides into a new strand
of DNA works only in the 3' to 5' direction. Consequently DNA synthesis only
occurs in the 5' to 3' direction. This means that one daughter strand can be made as
continuous strand but the other is made in short pieces that are linked together with
DNA ligase. The method is fast and accurate, only about one DNA nucleotide per
million is incorrectly paired. Errors are called mutations which can be passed on to
the next generation with good and bad consequences. Polymerase can proof read
new strands. If errors are detected the enzyme backs up, removes the incorrect
nucleotide and replaces it with a correct one. At the completion of the process 2
DNA molecules have been formed each identical to the original. One strand of
each of the new DNA molecules is a strand of the original DNA and the other
strand is the complementary strand made during replication. This is semiconservative replication.
Expression of Genotype
Small sections of chromosomes are genes. One’s genetic makeup is their genotype.
What is expressed, the specific traits is the phenotype. Phenotype is the result of
proteins. Proteins to be made are dictated by DNA. Genes do not directly make
proteins. They must transfer the information they have about specific proteins to
another nucleic acid-RNA or ribonucleic acid. This takes place in two processes:
transcription and translation. DNA directs ribonucleic acid synthesis. RNA is
made of monomers or nucleotides called ribonucleotides. These have the same
basic components as DNA: a 5 C sugar-ribose, phosphate groups and nitrogenous
bases. The bases are the same as in DNA with one exception. RNA has Uracil (U)
instead of T. The rules for base pairing are the same. Uracil is substituted for
thymine. So U-A not T-A.
Transcription transfers genetic information from DNA to RNA. That information
is used to make proteins via translation. There are 3 main types of RNA:
messenger (mRNA), ribosomal (rRNA) and transfer (tRNA). All are involved in
translation.
Genetic Code
DNAmRNAproteins; tRNA and rRNA are also involved. Proteins are long
strands of amino acids held by peptide bonds. There are from 50-27,000 amino
acids in each polypeptide. Each protein has a unique amino acid sequence. The
language of DNA is chemical. DNA must be translated into a different chemical
language, that of polypeptides. DNA language is written in the linear sequence of
nucleotide bases that comprise it. A specific sequence of bases makes up a gene.
One geneone protein.
During transcription DNAmRNA. The nucleic acid language of DNA is
rewritten as a sequence of RNA bases. This is still nucleic acid language.
Translation is the conversion of nucleic acid language into polypeptide language.
Polypeptides are macromolecules. They are polymer sof amino acids (monomers).
20 amino acids are common to all organisms. The sequence of nucleotides in
mRNA dictates the sequence of amino acids in the polypeptide. The mRNA’s
sequence is determined by DNA. The sequence of bases in a molecule of DNA is
the genetic code.
DNA and RNA are made of 4 different nucleotides and there are 20 amino acids. If
each nucleotide coded for one amino acid there could only be 4 amino acids. If
each 2 coded for one there could be 16 amino acids. The smallest number of bases
that can code for 20 amino acids is 3. A particular triplet of nucleotides in mRNA
is a codon which is specific for one particular amino acid. There are 64 possible
triplet codes. The code is redundant because there are more than one codon for
each amino acid. The first codon was deciphered in 1961 when Hirenberg made an
artificial RNA by linking 3 UspolyU. He put poly U into a test tube with
ribosomes and the ingredients for protein synthesis an obtained a polypeptide of
PHE or phenylalanine. Today all codons have been deciphered. 61 code for amino
acids. Some codons have regulatory purposes for example to start and stop. AUG
is the start codon and codes for MET-methionine. UAA, UAG, UGA are stop
codons. They tell ribosomes to end polypeptide synthesis.
The genetic code is highly conserved. It is the same in all organisms. Genes can be
transcribed and translated even if transferred from one species into another even
bacteria and humans. This has opened the door for genetic recombinant technology
and genetic engineering.
Transcription
Transcription is the process of transferring genetic information from DNA to
RNA. It is similar to DNA replication. DNA is used as a template to make RNA.
During transcription the 2 stands of DNA must separate. Only one serves as a
template (both are used in replication). Nucleotides take their places one at time
along the template using the same base pairing rules as for replication except A-U.
Transcription has 3 stages: initiation, elongation and termination. Initiation is the
first phase of trancription. RNA polymerase attaches to the promoter area, a
specific nucleotide sequence and RNA synthesis begins. RNA polymerase decides
which strand to use as the template. The strand used is the antisense strand.
Elongation is the second stage of transcription. During this stage the RNA strand
grows longer. The RNA strand peels away from the template allowing the
separated DNA strands to come back together. The RNA strand formed is directly
complementary to its DNA template; each time C is found in the antisense strand
of the DNA template a G is paired with it. The last stage of transcription is
termination. RNA polymerase reaches a special sequence of bases in the template
called the terminator which ends transcription and RNA polymerase detaches.
In prokaryotic cells RNA can function immediately. In eukaryotes RNA is
processed before moving to the cytoplasm for translation. There are several posttranscriptional modifications. One modification is capping-tailing. In this
procedure more nucleotides are added to either end of the RNA. A “G” nucleotide
is added to one end and 50-250 A nucleotides are added to the other. These
additions make the RNA molecule more stable. The ends protect the molecule
from attack by enzymes and helps ribosomes recognize mRNA.
Another modification is splicing and ligation. Precursor mRNA contains exons &
introns. Segments containing information for the formation of proteins are exons.
Introns are internal non-coding regions. Before mRNA can leave the nucleus the
introns must be removed from the strand. Introns are spliced out and exons are
ligated (or attached) together. Now the RNA can move to the cytoplasm through
nuclear membrane pores
Translation
Translation requires an interpreter, an intermediate that can understand the
language of one form and translate that message into another. The message of
DNA is transferred to an RNA molecule by transcription. This message needs to
translated into a protein. This process requires ribosomes, ATP and tRNA. tRNA
(transfer RNA) is an interpreter which converts the 3 letter code of nucleic acids
into amino acids. Each tRNA has a sequence of 3 nucleotides called an anticodon
which can bind, following the rules of bases pairing, complementary triplets of
nucleotides in the codon of the mRNA.
Cells ready to carry out translation have a supply of amino acids in their
cytoplasm. Amino acids are not able to recognize codons of mRNA on their own.
tRNA matches amino acids to their appropriate codons on mRNA. In order to do
this tRNA must pick the appropriate amino acid and recognize the appropriate
codon in the mRNA. The structure of tRNA allows it to perform these tasks. tRNA
is composed of one strand of RNA. The chain twists and folds on itself making
some double stranded areas. At one end is a special triplet of bases called the
anticodon. The anticodon contains a complementary sequence of bases to the
sequence of bases in the mRNA molecule. The anticodon recognizes the bases in
mRNA by applying the standard base pairing rules. At the other end of the tRNA is
a site when an amino acid can attach. An enzyme recognizes both the tRNA and its
amino acid partner and links the 2 using ATP. There are at least 32 different tRNA
in eukaryotic cells. Anticodons are redundant. There is at least one anticodon for
each amino acid.
Ribosomes coordinate the process of translation. Ribosomes are formed from 2
subunits each made of proteins and rRNA (ribosomal RNA). A completely
assembled ribosome has a binding site for mRNA on its small subunit and two
binding sites for tRNA on its large subunit. Translation has the same 3 stages as
transcription, that is initiation, elongation and termination.
Initiation begins when an mRNA molecules binds to a small ribosomal subunit. A
special initiator tRNA binds to the specific codon-AUG. This is the start codon.
The anticodon is UAC. The start codon also carries the amino acid methionine.
Next a large ribosomal subunit binds to a small one creating a functional ribosome.
The initiator tRNA fits into one of the two tRNA binding sites on the ribosome
called the P site. The other tRNA binding site, the A site is vacant. The P site holds
the tRNA containing the growing peptide chain and the A site holds the tRNA
carrying the next amino acid to be added to the chain. Once initiation is completed
elongation begins. In this process amino acids are added one by one to the first
amino acid. Each addition is composed of 3 steps. First the anticodon of an
incoming tRNA carrying an amino acid pairs with the mRNA codon in the A site
of the ribosome. Next a peptide bond forms between the carboxyl group of one
amino acid and the amino group of the next. To do this the polypeptide leaves the
tRNA in the P site and attaches to the amino acid on the tRNA in the A site. These
are attached by a peptide bond. The ribosome catalyzes bond formation.
The last stage of elongation is translocation. The P site tRNA leaves the ribosome
and the ribosome moves or translocates the tRNA in the A site with its attached
polypeptide to the P site. This movement brings the next mRNA codon to be
translated into the A site and the process begins again. Elongation continues until a
stop codon is reached. UAA, UAG and UGA are stop codons. This is the
termination stage of translation. The completed peptide is released from the last
tRNA and leaves the ribosome which splits back into its separate subunits.
A single mRNA has many ribosomes traveling along it called polysomes which are
in various stages of synthesizing polypeptide.
Mutations
Mutations are the result of any change in the nucleotide sequence of DNA. The
production of mutations is called mutagenesis and can occur in many ways. Some
mutations are spontaneous and occur do to mistakes in transcription or translation.
Other sources of mutants are radiation, chemicals and viruses. These are termed
mutagens.
Mutations can be sorted into two categories: base substitutions and base insertions
and deletions. Base substitutions refer to replacement of one nucleotide with
another and are called point mutations. A change in a single gene may go
unnoticed, improve the organism or may cause significant trouble. Sometimes a
substitution improves the protein for which it codes and enhances the success of
the individual into which it was placed. Since the genetic code is redundant
sometimes a substitution does not change the amino acid made. Many disorders are
the consequence of point mutations such as hemophilia, sickle cell anemia,
Huntingtons Chorea, and Tay Sachs disease.
Insertion and deletion mutations often have disastrous effects. mRNA is read as a
series of triplet codons during translation. Adding or deleting one base will change
the reading frame for tRNA. Frame-shift mutations have dramatic effects. All
nucleotides downstream from the insertion or deletion will be regrouped into
different codons and the result is usually a nonfunctional protein.
Mutations are rare. Every day 5000 A & G bases are lost and 100 cytosine bases
are damaged in any cell just from thermal disruption. Stability of genes and
therefore low mutation rate is due to efficient DNA repair mechanisms.