DNA and Protein.
The outstanding accomplishment of the 20th century, has been the discovery of DNA,
and its relationship to protein synthesis.
Whether an organism is a human being or a mouse, has black or blue eyes, has light or dark skin,
is determined by the proteins it possesses.
Moreover, within an individual organism, muscle cells differ from nerve cells or any other type of cell,
because of the types of protein they contain, and the functions performed by the proteins.
Crucial for an understanding of protein functions is the fact that each protein has a unique shape,
that determines its interaction with other molecules.
Since the functioning of the human body is determined primarily by its proteins,
hereditary information consists of a set of instructions, coded into DNA molecules,
that specify the types of proteins a cell can synthesise.
Different cell types have different proteins and the specifications for these proteins,
are coded in the DNA.
All the cells in the body, with the exception of sperm or ova, receive the same genetic information,
when DNA molecules are duplicated and passed on to the daughter cells, at the time of cell division.
Cells differ in structure and function because only a portion of the total genetic information,
common to all cells is used by any given cell to synthesise proteins.
In other words, different portions of the inherited genetic information,
are translated in different types of cells.
Protein binding sites.
Binding site characteristics.
The ability to bind various molecules and ions to specific sites on the surface of a protein molecule,
forms the basis for the wide variety of functions performed by proteins.
A ligand is any molecule or ion that is bound to the surface of a protein,
by forces other than covalent chemical bonds.
These forces are either
1. Attractions between oppositely charged ionic or polarised groups on the ligand and protein or,
2. The weaker attractions, due to Van der Waals forces, between adjacent non polar regions, on the two molecules.
The region of a protein to which a ligand binds is known as a binding site.
A protein may contain several binding sites, each specific for a different ligand.
Chemical specificity.
The force of electrical attraction between oppositely charged regions on a protein and a ligand,
decreases markedly as the distance between them increases.
The even weaker Van der Waals forces act only between non polar groups,
that are very close to each other.
Therefore in order for a ligand to bind to the surface of a protein, it must be close to the protein.
This occurs when the shape of the ligand is complementary to the shape of the protein binding site,
such that the two fit together like pieces of a jigsaw puzzle.
The binding between a ligand and a protein may also be so specific,
that a binding site may bind only one type of organic molecule or ion and no other.
Such selectivity allows a protein to ‘identify’, by binding,
one particular molecule in a solution containing hundreds of different molecules.
This ability of a protein to bind specific ligands is known as chemical specificity,
since the shape of the binding site determines the type of chemical compound that is bound.
The conformation of a protein is determined by the location of various amino acids,
along the polypeptide chain.
Accordingly, proteins with different amino acid sequences have different shapes,
and therefore different binding sites, each with its own chemical specificity.
The amino acids that interact with the ligand need not be adjacent to each other,
along the polypeptide chain,
since the folding of the protein may bring various segments of the molecule into juxtaposition.
Some binding sites have chemical specificity that allow them to bind only one type of ligand,
whereas other binding sites may be less specific, and thus be able to bind a number of related ligands.
Affinity.
The strength of a ligand - protein is a property of the binding site known as affinity.
The affinity of a binding site for a ligand determines how likely it is that a bound ligand,
will leave the protein surface and return to the unbound state.
Binding sites that tightly bind a ligand are called high affinity binding sites.
Affinity and chemical specificity, are two distinct, although sometimes related,
properties of binding sites.
Chemical specificity depends only on the shape of the binding site,
whereas affinity depends on the strength of the attraction between the protein and the ligand.
Thus, different proteins may be able to bind the same ligand, that is,
may have the same chemical specificity, but may have different affinities for it.
For example, a ligand may have a negatively charged ionised group that will bind strongly to a site,
containing a positively charged amino acid side chain, but would bind less strongly to a binding site,
having the same shape but no positive charge.
In addition, the closer the surfaces of a ligand and binding site to each other,
the stronger the electrical interactions.
Hence, the more closely the ligand shape matches the binding site shape, the greater the affinity.
In other words, shape can influence affinity as well as chemical specificity.
Saturation.
In a solution containing both ligands and binding sites,
some ligands will bind to unoccupied binding sites,
while some of the bound ligands will be coming off binding sites.
At any given time, a binding site will either be occupied or not occupied.
The term saturation refers to the fraction of binding sites that are occupied at any given time.
When all the binding sites are occupied,
the population of the binding sites is said to be 100% saturated.
When half of the available sites are occupied, the system is 50% saturated,
if it were occupied by a ligand, 50% of the time.
The degree of saturation of a binding site depends on two factors:
1. The concentration of unbound ligand in the solution and,
2. The affinity of the binding site for the ligand.
The greater the ligand concentration, the greater the probability of a ligand,
encountering an unoccupied binding site and become bound.
Thus, the percent saturation of binding sites will increase with increasing ligand concentration,
until all the sites become occupied.
If a ligand were a molecule that exerted a biological effect when it was bound to a protein,
the magnitude of the effect would increase with increasing number of bound ligands,
until all the binding sites were occupied.
Further increase in ligand concentration would produce no further effect,
since there would be no additional sites to be occupied.
To generalise, a continuous increase in the magnitude of a chemical stimulus (ligand concentration),
which exerts its influence by binding to proteins, will produce an increased biological response,
up to the point where the protein binding sites become saturated.
The second factor determining the degree of binding site saturation, is the affinity of the binding site.
Collisions between molecules in a solution, and those on a binding site,
can dislodge a loosely bound ligand.
If a binding site has a high affinity for a ligand, even a low ligand concentration,
will result in a high degree of saturation, since once bound to the site, the ligand is not easily dislodged.
A low affinity site, on the other hand, requires a much higher concentration of ligand,
to achieve the same degree of saturation,
since any given ligand remains bound for a much shorter time,
requiring more frequent encounters between unbound ligand and the binding site,
to keep the site occupied.
One measure of binding site affinity is the ligand concentration necessary,
to produce fifty percent saturation.
The lower the ligand concentration at 50% saturation, the greater the affinity of the binding site.
Competition.
More than one type of ligand can bind to certain binding sites.
Competition occurs when two or more ligands compete for the same binding site.
The presence of multiple ligands to bind to the same binding site,
affects the percentage of binding sites occupied by any one ligand.
If two competing ligands, A and B, are present, increasing the concentration of A,
will increase the amount of A that is bound,
and also decrease the number of sites available for the binding of B,
thus decreasing the amount of B that is bound.
As a result of competition, the biological effects of one ligand,
may be markedly diminished by the presence of another.
For example, many drugs produce their effects by competing with the body’s natural ligands,
for binding sites.
By occupying the binding sites, the drug decreases the amount of natural ligand that can be bound.
Regulation of binding site characteristics.
Because proteins are associated with practically every cell function,
the mechanism for controlling these functions, centre on the control of protein activity.
There are two ways to control protein activity:
1. Changing protein shape alters its binding of ligands.
2. Regulation of protein synthesis determines the amounts of protein in a cell.
The first type of regulation - control of protein shape will be discussed in this module.
Since protein shape depends on electrical attractions between charged or polarised groups,
in various regions of a protein, a change in the charge distribution along a protein,
or in the polarity of the molecules immediately surrounding it, will alter its shape
Two non specific factors that alter protein shape are temperature,
which alters the amount of kinetic energy available for breaking the electrostatic attractions,
between various regions of a protein, and acidity, which alters the ionisation of amino acid side chains.
Because both of these factors are maintained relatively constant in the body,
they are not used to selectively regulate protein activity.
Should large changes in temperature or acidity occur however, as in disease,
they can produce market alterations in protein shape and thus in protein activity.
The two mechanisms that are used by sense to selectively alter protein shape,
are allosteric and covalent modulation.
Allosteric modulation.
Whenever a ligand binds to a protein, the attracting force between the ligand and the protein,
alters the protein’s shape.
Very importantly, ligand binding can change the shape of the protein,
in regions that are not part of the binding site.
Therefore, when a protein contains two binding sites,
the binding of ligand to one site can alter the shape of the second binding site,
and hence the binding characteristics of that site.
This is called allosteric (other shape) modulation, and such proteins are called allosteric proteins.
One binding site on a allosteric protein, known as the functional site,
carries out the protein’s physiological function.
The other binding site is the regulatory site, and the ligand that binds with this site,
is known as a modulator molecule, since its binding to the regulatory site alter the shape,
and thus the activity of the functional site.
The regulatory site to which modulator molecules bind is the equivalent of a molecular switch,
that controls the affinity of the functional site.
In some allosteric proteins, the binding of the modulator molecule to the regulatory site,
turns on the functional site by changing its shape, so that it can bind and shape the functional ligand.
In other cases, the binding of a modulator molecule turns off the functional site,
preventing the functional site from binding its ligand, a state of zero affinity.
In still other cases, binding of the modulator molecule may decrease or increase,
the affinity of a functional site, so that the percentage saturation of the functional site,
will be decreased or increased, at any given concentration of the ligand,
that binds to the functional site.
A single cell contains not just one but many identical molecules of any particular allosteric protein.
The total activity of these proteins depends on how many of the regulatory sites,
are occupied by modulator molecules.
Therefore, the magnitude of a cell function can be regulated by varying the concentration,
of the modulator molecule, and thus the number of allosteric proteins,
that have been altered by binding modulator molecules.
It should be emphasised that most proteins are not subject to allosteric modulation.
Only certain key proteins associated with specific cell functions are regulated.
Covalent modulation.
A second wave to alter the shape and therefore the activity of a protein,
is to covalently bind charged chemical groups to some of the protein’s side chains.
This is known as covalent modulation.
In most cases a phosphate group, which has a net negative charge,
is covalently attached by a chemical reaction called phosphorylation.
When one of the amino acids side chains in a protein is phosphorylated,
a negative charge is introduced into this region of the protein.
This alters the distribution of electrical forces in the protein,
producing a change in protein conformation.
If this conformational change occurs in the region of a binding site,
it will change the binding site’s affinity.
The effects produced by covalent modulation are the same as those of allosteric modulation,
that is, phosphorylation may turn a binding site on or off, or alter its affinity for a ligand.
Unlike allosteric modulation, which involves simple binding of modulator molecules,
covalent modulation requires chemical reactions in which covalent bonds are formed.
Such reactions are mediated by a special class of proteins known as enzymes.
Enzymes accelerate the rate at which one or more initial molecules,
called substrates are converted to new molecules called products.
Any enzyme that mediates protein phosphorylation is called a protein kinase.
These enzymes catalyse the transfer of phosphate from a molecule of adenosine triphosphate (ATP),
to a hydroxyl group present on the side chain of certain amino acids.
Protein + ATP Protein kinase ——> protein-PO42- + ADP.
This can be read as protein + ATP in the presence of protein kinase results in a protein phosphate,
with a charge of minus 2 + ADP.
The protein and ATP are the substrates for protein kinase,
and phosphorylated protein and ADP are the products of the reaction.
There is also a mechanism for moving the phosphate group and returning the protein,
to its original shape.
This dephosphorylation is accomplished by a second enzyme known as phosphoprotein phosphatase.
Protein-PO42- + H2O phosphoprotein phosphatase —> protein + HPO42-.
This can be read as protein phosphate with a charge of minus 2, + H 2 O,
in the presence of phosphoprotein phosphatase results in a protein + HPO four with a charge of minus 2.
Thus, two enzymes control a protein’s activity by covalent modulation, one that adds phosphate,
and one that removes phosphate.
Although most proteins contain amino acids capable of being phosphorylated,
only those proteins that are substrates for specific protein kinases are phosphorylated.
There are many protein kinases, each with specificities for different proteins,
and several might be present in the same cell.
The chemical specificities of the phosphoprotein phosphatases are broader,
and a single enzyme can dephosphorylate many different phosphorylated proteins.
Moreover, unlike protein kinases, phosphoprotein phosphatases tend to be continuously active.
When a protein kinase is active, the rate of phosphorylation exceeds that of dephosphorylation,
and most of the proteins being covalently modulated are maintained in the phosphorylated state.
Dephosphorylating such proteins require only stopping the kinase reaction,
since the continuously active phosphoprotein phosphate will remove the phosphate groups.
The kinase reaction can be controlled by modulator molecules,
since the protein kinases are allosteric proteins.
Thus, the process of covalent modulation is itself regulated by allosteric mechanisms.
Genetic information and protein synthesis.
Genetic Information.
Molecules of DNA contain instructions, coded in the sequence of nucleotides,
for the synthesis of proteins.
There are many molecules of DNA in the cell nucleus, each containing different set of instructions.
A sequence of nucleotides containing the information that determines the amino acid sequence,
of a single polypeptide chain, is known as a gene.
Genes are units of hereditary information, and there are about 20,000 protein coding genes.
As an example, of the expression of genetic information, consider eye colour,
which is due to the presence of pigment molecules in certain cells of the eye.
A sequence of enzyme mediated reactions is required to synthesise these pigments,
and the genes that determine eye colour do so by controlling the synthesis,
of the enzymes in this pathway.
These genes do not contain any information about the chemical structure of the pigments.
All they contain is the information required to synthesise the enzymes,
that mediate the formation of the pigments.
Although DNA contains the information required for the synthesis of proteins,
it does not itself participate directly in the assembly of protein molecules.
Most of a cell’s DNA is in the nucleus.
A small amount is present in the mitochondria.
Most protein synthesis occurs in the cytoplasm.
The transfer of information from DNA to the site of protein synthesis is the function of RNA molecules,
whose synthesis is governed by the information in DNA.
The mechanism for expressing genetic information occurs in all living organisms.
A molecule of DNA consists of two polynucleotide chains coiled around each other,
to form a double helix .
Each nucleotide contains one of the four bases - adenine (A), guanine (G), cytosine (C) or thymine (T).
Each of these bases is specifically paired,
A to T,
G to C,
with the base of the opposite chain of the double helix.
Thus, both nucleotide chains contain a specifically ordered sequence of bases,
one chain being complementary to the other.
The genetic language is similar in principle to a written language, which consists of a set of symbols,
forming an alphabet.
The letters are arranged in specific sequence to form words, and the words are arranged in linear
sequence to form sentences.
The genetic language contains only four letters, corresponding to the four bases A,G,C, and T.
The words are three - base sequences that specify particular amino acids.
Each word in the genetic language is only three characters long.
This is termed a triplet code.
These words are arranged in a linear sequence along DNA,
and the sequence of words specifying the structure of a single protein makes up a gene.
A typical human gene is a sequence of approximately 20,000 base pairs.
Thus, in our analogy, a gene is equal to a sentence.
The entire collection of genes in a cell is equivalent to a book.
Since the four bases can be arranged in 64 different letter combinations (4 into 4 into 4 = 64),
a triplet actually provides more than enough code words.
It turns out that not just 20, but 61 of the 64 possible triplets are used to specify amino acids.
This means that a given amino acid is usually specified by more than one code word.
For example, the four triplets C-C-A, C-C-G, C-C-T and C-C-C, all specify the same amino acid, glycine.
The three triplets that do not specify amino acids are known as termination code words.
They perform the same function as does a period at the end of the sentence.
They indicate that the end of a genetic message has been reached.
The code word for the amino acid methionine does double duty,
by also indicating the beginning of a protein chain.
The genetic code is a universal language used by all living cells.
For example, the code words for the amino acid tryptophan are the same in the DNA of a bacteria,
and amoeba, a plant and a human being.
Although the same code words are used by all living cells, the sequences of code words (the sentences),
that determines the amino acid sequences in proteins, are different in each organism.
The universal nature of the genetic code supports the concept,
that all forms of life on Earth evolved from a common ancestor.
Protein synthesis.
SNA molecules are too large to pass through the nuclear membrane into the cytoplasm.
It is in the cytoplasm, on ribosomes, that the proteins are synthesised.
Since DNA cannot leave the nucleus, a message carrying genetic information,
must pass from the nucleus to the cytoplasm.
This message is carried by the RNA molecules known as messenger RNA, (mRNA).
The transfer of genetic information from DNA to protein thus occurs in 2 stages.
First, the genetic message is passed from DNA to mRNA in the nucleus (transcription).
Second, the message in mRNA passes from the nucleus into the cytoplasm,
where it is used to direct the assembly of the specific sequence of amino acids,
to form a protein (translation).
Transcription: mRNA synthesis.
During transcription, the sequence of nucleotides in mRNA.
Ribonucleic acids are single chain polynucleotides, whose nucleotides differ from DNA,
in that they contain the sugar ribose (rather than deoxyribose ) ,
and the base uracil (rather than thymine).
The other bases adenine, guanine, and cytosine - occur in both DNA and RNA.
The poll of subunits used to synthesis mRNA are free (uncombined) ribonucleotides,
each containing 3 phosphate groups (nucleotide triphosphate): ATP, GTP, CTP, and UTP.
In DNA, the two poly nucleotide chains are linked together by hydrogen bonds,
between specific pairs of bases - A pairing with T, and G with C.
Transcription begins with the breakage of these hydrogen bonds,
so that a portion of the two chains of the DNA double helix separates.
The bases in the exposed DNA nucleotides are then able to pair with the bases,
in the free ribonucleotide triphosphate.
Free ribonucleotide containing adenine pair with any exposed thymine base in DNA.
Like wise, free ribonucleotide containing G, C or U pair with an exposed C. G and A, respectively.
Uracil, which is present in RNA but not in DNA pairs with the base adenine in DNA.
In this way, the nucleotide sequence in DNA acts as a template,
that determines the sequence of nucleotides in mRNA.
The aligned nucleotides are joined together by the enzyme RNA polymerase.
This enzyme, which binds to the DNA, catalyses both the splitting-off,
of two of the three phosphate groups from each nucleotide and the covalent linkage,
of the RNA nucleotide to the next one in sequence.
RNA polymerase is active only when bound to DNA and will not link free nucleotides together,
in what would be a random sequence, when they are not base paired with DNA.
DNA acts as a modulator molecule that allosterically activates RNA polymerase.
Since DNA consists of two strands of polynucleotides, both of which are exposed during transcription,
it should theoretically be possible to form to different mRNA molecules, one from each strand.
However, only one of the two potential mRNAs is ever formed.
Which of the two DNA strands is used as a template for mRNA synthesis,
is determined by a specific sequence of nucleotides in DNA, called the promoter,
located at the beginning of each gene.
The promoter, to which RNA polymerase binds, is present in only one of the two DNA strands.
Beginning at the promoter end of a gene, the RNA polymerase separates the two DNA strands,
as it moves along one strand, joining one ribonucleotide at a time to the growing mRNA chain,
until it reaches a termination code word at the end of the gene.
This causes the RNA polymerase to release the newly formed mRNA.
In a given cell, the information in only few of the thousands of genes present in DNA,
is transcribed into mRNA at any given time.
Genes are transcribed only when RNA polymerase can bind to their promoter sites.
Various mechanisms are used by cells either to block or to make accessible the promoter region,
of any particular gene.
Such regulation of gene transcription provides a means of controlling the synthesis of specific proteins, and thereby the activities of the cell.
The nucleotide sequence in mRNA is not identical to that in the corresponding strand of DNA,
since its formation depends on the pairing between complementary, not identical, bases.
A 3 base sequence in mRNA that specifies one amino acid is called codon.
Each codon is complementary to a 3 base sequence in DNA.
For example, the base sequence T-A-C in DNA corresponds to the codon A-U-G in mRNA.
Although the entire sequence of nucleotides in a gene is transcribed,
into a corresponding sequence of nucleotides in mRNA,
only selected portions of this sequence code for sequences of amino acids.
These nucleotides sequences, known as exons,
are separated from each other by non coding sequences of nucleotides known as introns.
Before passing to the cytoplasm, a newly formed mRNA must undergo RNA processing,
to remove the intron sequences.
Nuclear enzymes that identify specific nucleotide sequences at the beginning and end of each introns,
remove the introns and splice the end of one exon to the beginning of another exon,
to form an mRNA with no intron segments.
In some cases, the exons derived from a single gene can be sliced together in several different sequences,
resulting in the formation of different mRNAs from the same gene and giving rise, in turn,
to several different proteins.
The mRNAs formed as a result of RNA processing are 75 to 90% shorter,
than the original transcribed mRNA,
meaning that 75 to 90% of the nucleotide sequences in DNA are introns.
What role that such large amounts of ’nonsense’ DNA may perform is unclear.
In addition to genes that give rise to mRNA, and hence code for proteins,
some genes form other types of RNA.
Translation: polypeptide synthesis.
Once transcribed and processed, mRNA moves through the pores in the nuclear envelope,
into the cytoplasm, where it binds to a ribosome.
Ribosome is the cell organelle that contains the enzymes an other components required for translation of mRNA’s coded message into a protein.
Before describing the assembly process, we must present the structure of a ribosome,
as well as the characteristics of two additional types of RNA, involved in protein synthesis.
Ribosomes.
Ribosomes are small granules, with a diameter of about 23 nanometres, located in the cytoplasm.
It is either suspended in the cytosol (free ribosomes) are attached to the surface,
of the endoplasmic reticulum (bound ribosomes).
Protein synthesised on free ribosomes are released into the cytosol.
Protein synthesised on bound ribosomes are released into the lumen of the endoplasmic reticulum,
from which they are either secreted from the cell or transferred to various organelles.
Each ribosome consists of two subunits, and large 60S and the small 40S subunit.
These subunits contain many proteins in association with the type of RNA known as ribosomal RNA(rRNA).
Ribosomal RNA is synthesised in the nucleus, with DNA again serving as a template,
for positioning the sequence of nucleotides in rRNA.
The genes that code for rRNA are associated with the nucleolus,
which is also the site at which the components of the two subunits of a ribosome are assembled.
The ribosomal subunits then move into the cytoplasm.
When an mRNA molecule arrives in the cytoplasm, one end of it binds to the 40S subunit,
and then this combination binds to the 60S subunit to form a fully functional ribosome,
with the portion of the RNA lying in a groove between the subunits.
The mRNA is now ready to direct protein assembly.
The sequence of codons in mRNA specifies the order of amino acids in the protein.
Transfer RNA.
How do individual amino acids identify the appropriate codons in mRNA during the process of translation?
By themselves, free amino acids do not have the ability to bind to the bases in mRNA codons.
This process of identification involves a third type of RNA, known as transfer RNA, tRNA.
Transfer RNA molecules are the smallest, (with about 80 nucleotides long), of the three types of RNA.
The single chain of tRNA looks back upon itself,
forming a structure resembling a clover leaf with three loops.
Like mRNA and rRNA, tRNA is synthesised in the nucleus, by base pairing with DNA nucleotides,
in this case at specific tRNA genes, and then moves to the cytoplasm.
The key to tRNA’s role in protein synthesis is that it can combine with the specific amino acid,
and a codon in mRNA specific for that amino acid.
Transfer RNA is covalently linked to an amino acid by an enzyme known as aminoacyl -tRNA synthetase.
There are at least 20 different aminoacyl -tRNA synthetases,
each of which catalysis the linkage of a specific amino acid to a particular type of tRNA.
The next step is to link the tRNA, bearing its attached amino acid, to the mRNA code of the amino acid.
This is achieved by base pairing between the tRNA and mRNA.
A 3 nucleotide sequence at the end of one of the loops of tRNA,
can base pair with a complimentary codon in mRNA.
This tRNA triplet sequence is appropriately termed as an anti codon.
For example, in the amino acid alanine, the tRNA is covalently linked to alanine at one end,
and that its anti codon C-G-U is base paired with the codon G-C-A in mRNA at the other end.
Protein assembly.
The individual amino acids linked to mRNA by tRNA must now be bound to each other by peptide bonds.
Several ribosomal proteins and rRNA interact in a complex manner to identify the initial codon’s sequence in mRNA, and to catalyse the formation of a peptide bond between the first and second amino acids,
in the peptide chain being synthesised.
This initial step is the slowest step in protein assembly,
and the rate of protein synthesis can be regulated by factors that influence this initiation process.
Following the initiation of a protein’s synthesis,
the polypeptide chain is elongated by the successive addition of amino acids.
The 60S subunit of the ribosome has two binding sites for tRNA.
One holds the tRNA that is attached to the most recently added amino acid,
and the other holds the tRNA containing the next amino acid to be added to the chain.
Ribosomal enzymes catalysis the formation of a peptide bond between these two amino acids.
Following the formation of the peptide bond, the tRNA at the first binding site,
is released from the ribosome, and the tRNA at the second site - now linked to the peptide chain -
is transferred to the first binding site.
The ribosome moves one codon space along the mRNA,
making room for the binding of the next amino acid-tRNA molecule.
This process is repeated over and over,
as each amino acid is added in succession to the growing peptide chain,
at an average rate of 2 to 3 amino acids per second.
When the ribosome reaches the termination code in mRNA, specifying the end of the protein,
the link between the polypeptide chain and the last tRNA is broken,
and the completed protein is released from the ribosome.
The same strand of mRNA can be used to synthesise many molecules of the protein,
because the message in mRNA is not destroyed during protein assembly.
While one ribosome is moving along a strand of mRNA,
a second ribosome may become attached to mRNA, and begin the synthesis, of a second protein molecule.
Thus, a number of ribosome, as many as 70, may be attached to the same strand of mRNA,
at any one time.
Ribosomes at the beginning of mRNA have short peptide chains,
representing the first few amino acids in the protein.
Ribosomes near the end have protein chains that are almost completed.
Molecules of mRNA do not remain in the cytoplasm indefinitely.
Eventually they are broken down into nucleotides by cytoplasmic enzymes.
Therefore, if a gene corresponding to particular protein ceases to be transcribed into mRNA,
the synthesis of that protein will eventually slow down and cease, as the mRNA is broken down.
Changes can occur in the structure of some polypeptide chains following their synthesis on a ribosome.
Certain classes of proteins, the glycoproteins for example, are found by the post translational addition,
of various carbohydrate groups to the protein.
In other cases, specific peptide bond in a large polypeptide are broken down,
to produce a number of shorter polypeptides, each of which may perform a different function.
Steps from DNA to protein synthesis.
Transcription.
1. RNA polymerase binds to the promotor region of a gene,
and separates the two strands of the DNA double helix, in the region of the gene to be transcribed.
2. Free ribonucleotide triphosphate base-pair with the deoxynucleotides in DNA.
3. The ribonucleotides paired with one strand of DNA are linked by RNA polymerase to form mRNA,
containing a sequence of bases complimentary to one strand of the DNA base sequence.
4. mRNA processing removes the intron regions of mRNA, which contain noncoding sequences,
and splices together the exon regions, which code for specific amino acids.
Translation.
5. Processed mRNA passes from the nucleus to the cytoplasm,
where one end of the mRNA binds to the ribosome.
6. Free amino acids are linked to their corresponding tRNAs by aminoacyl-tRNA synthetase.
7. The three base anticodon in an amino acid-tRNA complex pairs with the corresponding codon,
in the region of the mRNA bound to the ribosome.
8. The portion of the peptide that has already been synthesised,
(and is still attached to the tRNA bound to the ribosome) is now linked by a peptide bond,
to the amino acid at the end of the tRNA next to it, thereby adding one more amino acid to the chain.
9. The tRNA that has been freed of the peptide chain, is released from the ribosome.
10. The ribosome moves one codon step along mRNA.
11. Steps from 7 to 10 are repeated over and over until the end of the mRNA message is reached.
12. The completed protein chain is released from the ribosome,
when the termination codon in mRNA is reached.
13. In some cases, the protein undergoes post translational processing,
in which various chemical groups are attached to specific side chains,
or the protein is split into several smaller peptide chains.
Replication and expression of genetic information.
The development of the human body from a single fertilised ovum involves cell growth, cell division,
and the differentiation of cells into specialised types such as nerve and muscle cells.
The maintenance of structure and function in the adult body also requires the same processes.
Each depends on the controlled synthesis of proteins.
Cell division requires, in addition, the replication of DNA and the transmission of identical copies,
of this genetic information to each of the two resulting cells.
Replication of DNA.
DNA is the only molecule in a cell able to duplicate itself,
without information from some other cell component.
In contrast, mRNA can be formed only in the presence of DNA,
protein can be formed only if mRNA is present,
and all other molecules synthesised by a cell are formed by metabolic path ways,
that use proteins in the form of enzymes.
The replication of DNA is, in principle, similar to the process when mRNA is synthesised.
During DNA replication, the two strands of the double helix separate,
and the exposed basis in each strand base-pair with free deoxyrebonucleotide triphosphate present,
in the nucleus.
The enzyme DNA polymerase then links the free nucleotides together forming a new strand of DNA,
a process very similar to that used to form mRNA.
In contrast to this synthesis of mRNA, where only one strand of DNA was used as a template,
here both strands of the original DNA act as templates for the synthesise of new strands.
The end result is two identical molecules of DNA, each called a ‘copy’.
In each copy, one strand of nucleotide was present in the original DNA molecule,
and one strand has been newly synthesised.
Prior to cell division, the DNA molecules in the nucleus are replicated by the above process,
and one copy will be passed on to each of the two new cells, termed daughter cell, when the cell divides.
Thus, the daughter cells receive the same set of instructions present in the parent cell.
Cell division.
Starting with the single fertilised egg cell, the first cell division produces two cells.
When these daughter cells divide, they each produce two cells, giving a total of 4 cells.
4 cells produce 8 cells, and so on.
Thus, starting from a single cell, three division cycles will produce 2 to the power of 3, which is 8 cells,
10 division cycles will produce 2 to the power of 10, which is 1024 cells,
and 20 division cycles will produce, 2 to the power of 20, which is 1,048,576 cells.
If the development of the human body involved only the cycle of cell division and growth,
without any cell death, it would only require 46 divisions to produce all the cells in the adult body.
However, large number of cells die during the course of development,
and even in an adult body many cells survive only for a few days,
and are continuously being replaced by the division of existing cells.
Aside from the morphological description of the various stages of the division process as visualised,
with a electron microscope, surprisingly little is known about the underlying molecular events,
that occur during division, or how the process is initiated and regulated.
Although the time between cell divisions varies considerably in different type of cells,
the most rapidly growing cells, divide once every 24 hours.
During most of this period there is no visible evidence that the cell will divide.
For example, in a 24 hour division cycle, visible changes in cell structure begin to appear,
23 hours after the previous division.
The period between the end of one division, and appearance of the structural changes,
that indicate the beginning of the next is known as interphase.
Since cell division takes only about 1 hour, the cell spends most of its time in interphase.
One very important event related to subsequent cell division does occur during interphase,
the replication of DNA, which begins 10 hours prior to the first visible signs of division.
Cell division involves 2 processes:
Nuclear division called mitosis and cytoplasmic division called cytokinesis.
Although mitosis and cytokinesis are 2 different events, the term mitosis is often used in a broad sense,
to include this subsequent cytokinesis.
Mitosis that is not followed by cytokinesis produces multinucleated cells formed in the liver,
placenta, and in some embryonic cells and cancer cells.
During most of the interphase, DNA is dispersed through out the nucleus in association with proteins,
to form 46 extended nucleoprotein threads known as chromatin.
The DNA in each chromatin thread has a different nucleotide sequence,
and therefore carries a different set of genes.
If a cell is to divide, the DNA of each chromatin thread replicates during interphase,
the result being 2 identical threads termed sister chromatids.
Each pair is joined together at one point known as the centromere.
As a cell enters mitosis, each chromatid pair becomes highly coiled and condensed,
forming rod shaped bodies known as chromosomes.
As the chromosomes condense, nuclear membrane breaks down,
and the chromosomes become linked, at their centromeres, to spindle fibres.
The spindle fibres, composed of microtubules, generate the forces then divide the cell.
Some of the spindle fibres extend between the two centrioles located on opposite sides of the cell,
while others are connected to the chromosomes.
Each centrioles consists of two micro tubular bodies, oriented at right angles to each other.
A single centrioles will pass on to each of the daughter cells during cytokinesis.
During the preceding interphase, at the time that DNA was replicated, the second centriole formed,
and the two centrioles moved to opposite sides of the nucleus.
The spindle fibres and centrioles constitute the mitotic apparatus.
As mitosis proceed, the sister chromatids in each chromosome separate at the centromere,
and move towards the opposite centrioles.
The spindle fibres act as though they were pulling the chromatids towards the poles.
Cytokinesis begins as the sister chromatids separate.
The cells begin to constrict along a plane perpendicular to the axis of the mitotic apparatus,
and constriction continues until the cell has been pinched in half, forming two daughter cells.
Following cytokinesis, the spindle fibres dissolve, a nuclear envelop forms,
and the chromatids uncoil in each daughter cell.
Cell differentiation.
Since identical sets of DNA molecules pass to each of the daughter cells during cell division,
every cell in the body, with a exception of reproductive cells,
contain the same genetic information as every other cell.
How, then is it possible for one cell to become a muscle cell and synthesise muscle proteins,
while another cell containing the same genetic information differentiates into a nerve cell,
and synthesises a different set of proteins?
This is because different combinations of genes are active in different cells.
The genes that contain the information for synthesis of muscle proteins synthesise mRNA in muscle cells.
The same genes are also present in nerve cells, but don’t form mRNA.
Other genes are active in nerve cells, but are not active in muscle cells.
The problem of cell differentiation is thus related to the general problem of regulating protein synthesis.
Certain genes are ‘turned on’ or ’turned off’ during cell differentiation.
The signals that control the transcription of specific genes are mostly unknown.
In addition, there is a problem of timing.
Some cells differentiate early during the embryonic development, others later, or even after birth.
What determines the timing of the signals that turn on certain genes,
and inhibit others during the course of development, is a subject still under research.
Mutation.
In order to form the 40 trillion cells of the adult human body,
a minimum of 40 trillion cell divisions must occur.
Thus, the DNA in the original fertilised ovum must be replicated at least 40 trillion times.
Actually, many more than 40 trillion divisions occur during the growth of a ovum,
into an adult human being, since many cells die during development,
and are replaced by the division of existing cells.
If a secretary were to type the same book 40 trillion times, one would expect some typing errors.
Therefore, it is not surprising to find that during the duplication of DNA,
errors occur that result in an altered sequence of bases, and a change in the genetic message.
What is amazing is that DNA can be duplicated so many times, with relatively few errors.
Any alterations in the genetic message carried by DNA is known as a mutation.
Factors in the environment that increase the mutation rate are known as mutagens,
and include certain chemicals and various forms of ionising radiation, such as x-rays,
cosmic rays, and atomic radiation.
Most of these mutagens break chemical bonds in DNA, so that incorrect pairing between bases occur,
are the wrong base is incorporated, when the broken bonds are reformed.
Even in the absence of specific agents that increase the likelihood of mutations,
mistakes in DNA copying do occur, so the mutation rate in never zero.
The simplest type of mutation occur when a single base is inserted in the wrong position in DNA.
For example, the base sequence C-G-T forms the DNA code for the amino acid alanine.
If guanine G is replaced by adenine A in the sequence, it becomes C-A-T,
which is the code for the amino acid valine.
It is during the replication of DNA, when free nucleotides are being incorporated into new strands of DNA,
that incorrect base substitution is likely to occur.
In a second type of mutation, large sections of DNA are deleted from the molecule,
or single bases are added and deleted.
Since the code is read in sequences of three bases,
the removal of one base not only alters the code word containing that base,
but also causes a misleading of all subsequent bases by shifting the reading sequence.
Addition of a single base would cause a similar misreading.
Such mutations will result either in no protein being formed, if the gene has been deleted,
or in the formation of a nonsense protein, a protein in which the amino acid sequence,
does not correspond to the functional protein, and single bases are added or deleted.
Assume that a mutation has altered a single code word, so that it now codes for a different amino acid,
say for example, alanine G-C-T to valine C-A-T.
What effect does this mutation have upon a the cell?
The answer depends upon both the type of gene and where the gene mutation has occurred.
Although proteins recomposed of many amino acids, the properties of a protein often depend upon,
only a very small region of the total molecule, such as the binding site of an enzyme.
If the mutation does not alter the conformation of the binding site,
there may be little or no change in the protein properties.
On the other hand, if the mutation alters the binding site,
a marked change in the protein’s properties may occur.
Thus, if the protein is an enzyme, a mutation may render it totally inactive,
or change its affinity for a substrate.
The mutated enzyme may even catalyse an entirely different type of reaction.
Let us assume that the mutation leads to an enzyme that is totally inactive.
If the enzyme is in the pathway supplying most of the cell’s chemical energy,
the loss of the enzyme may lead to death of the cell.
On the other hand, the normal enzyme may be involved in the synthesis of a particular amino acid,
and if the cell can obtain that amino acid from the extracellular fluid,
the cells functioning will not be impaired by the mutation.
To generalise, a mutation may have any of the three effects on the cell.
1. It may cause no noticeable change in the cell’s functioning.
2. It may modify cell function but still be compatible with cell growth and replication.
3. It may lead to cell death.
With one exception - cancer, the malfunction or death of a single cell, other than sperm or ovum,
as a result of mutation, usually has no significant effect, because there are so many cells,
performing the same operation in an organ.
Unfortunately, the story is more complex with the mutation has happened in a sperm or ovum,
since these cells combined to form the fertilised ovum from which all the cells of a new person will develop.
In this case the mutation will be passed on to all the cells in the body.
Thus, mutations in the ovum or sperm do not affect the individual in which they occur, but do affect,
often catastrophically, the children produced by these cells.
More over these mutations will be passed on to some individuals in future generations,
descended from the individual carrying the original mutant gene.
Inherited disease resulting from gene mutation are termed inborn errors in metabolism.
An example is phenylketonuria, a disorder that can lead to a form of mental retardation in children.
Because of a single abnormal enzyme, these persons are unable to convert the amino acid phenylalanine,
to the amino acid tyrosine at a normal rate.
Phenylalanine is therefore diverted into other biochemical pathways in large amounts,
giving rise to products that interfere with the normal activity of the nervous system.
These products are also excreted in large amounts in the urine, accounting for the name of the disease.
Fortunately, the symptoms of the disease can be prevented if the content of phenylalanine,
in the diet is restricted during childhood, thus preventing accumulation of the toxic products,
formed from phenylalanine.
Cell posses mechanism for protecting themselves from certain types of mutation.
For example, abnormal base pair occurs in DNA, such as C with T, (C normally pairs with G),
a particular set of enzymes will cut out the segment containing the abnormal base T,
allowing the normal strand to re synthesise the deleted segment by base pairing.
Mutations contribute to evolution.
Mutations may alter the activity of an enzyme in such a way that it is more, rather than less, active,
or they may introduce a new type of enzyme activity into the cell.
If an organism carrying such a mutant gene is able to perform some function more effectively,
than an organism lacking the mutant gene, it has the better choice of surviving,
and passing the mutant gene on to its descendants.
If the mutation produces an organism that functions less effectively than other organisms,
lacking the mutation, the organism is less likely to survive and pass on the mutant gene.
This is the principle of natural selection.
Although any one mutation, if it is able to survive in the population,
may cause only a very slight alteration in the properties of a cell, given enough time,
a large number of small changes can accumulate to produce very large changes,
in the structure and function of an organism.
Cancer.
In the adult body, the rate at which new cells in a tissue are formed, and old cells die are in balance,
producing a steady state, in which the tissue does not increase in size.
If the mechanism regulating cell division are altered, however,
the affected cells may produce new cells faster than old cells are removed,
forming a growing mass of tissue known as a tumour.
If the tumour cells remained localised and do not invade surrounding tissues,
it is said to be a benign tumour.
If, however the tumour cells grow into the surrounding tissues, disrupting their functions,
and/or spread to other regions of the body, it is said to be a malignant tumour,
and may lead to the death of the organism.
Malignant tumours are composed of cancer cells, which are cells that have lost the ability to respond,
to the normal control mechanisms, that regulate cell growth.
Cancer cells are characterised by their capacity for unlimited multiplication,
and for their ability to breakaway from the parent tumour, and spread by way of the circulatory system,
to other parts of the body, where they form multiple tumour sites, a process known as metastasis.
If cancer is detected in the early stages of its growth, before it has metastasised,
the tumour may be removed by surgery.
Once it has metastasised to many organs, surgery is no longer possible.
Drugs and radiation can be used to inhibit cell multiplication, and destroy malignant cells,
both before and after metastasis.
Unfortunately, these treatments also damage the growth of normal cells.
Any normal cell in the body may at some point undergo a transformation to a malignant cell.
A number of agents known as carcinogens, such as radiation, viruses, and certain chemicals,
can induce the cancerous transformation of cells.
These agents act by altering or activating various genes associated with cell growth.
Mutagenic agents also tend to be carcinogenic.
The mutation for certain genes in animal cells has been found to transform these cells into cancer cells,
and similar genes have been found in human cells.
Such cancer producing genes are called oncogenes.
Mutations produced by carcinogens are, however, only the first step in developing of many cancers.
In addition, chemical agents, such as hormones are environmental chemicals,
may be required to cause the enhanced cell replication, leading from a transformed cell to a tumour.
At present it is unclear whether a single mutation or two or more mutations are necessary to transform,
a normal cell into a cancer cell.
The multiple mutation hypothesis provides one explanation for the increased incidents of cancer with age.
Some individuals may inherit one oncogene and thus be at increased risk of developing cancer,
if they should undergo a second mutation.
The body’s defence system is normally able to detect and destroy most cancerous cells.
These defect systems become less efficient with age,
which also contributes to the increased incidents of cancer with age.
Little is currently known about the molecular mechanisms that are altered by oncogenes,
or the regulatory systems that control normal tissue growth.
Some of the proteins produced by oncogenes have been identified, however.
There are other protein kinases are signal detecting proteins that bind messenger molecules,
involved in regulating growth.
In some cases it appears that the gene, prior to mutation, produces a protein,
that inhibits the transcription of another gene involved in growth.
The loss of this inhibeitary protein through mutation,
allows the unrestrained transcription of the growth promoting gene.
Recombinant DNA.
In the early 1970’s, bacterial enzymes were discovered that split molecules of DNA in a unique manner.
These enzymes, called restriction nucleases, identify specific nucleotide sequences,
usually four to six nucleotides long, and in this region they cut DNA at a different site on each strand.
The cut strands each have short exposed sequence that are complement to each other,
and can bind together by base pairing.
A restriction nucleases breaks the DNA strand at many points, resulting in a number of DNA segments,
each having an exposed base sequence at each end, that can bind to the ends of other DNA segments, split by the same restriction nuclease.
If the DNA from two organisms is cut by the same restriction nuclease,
then a DNA fragment from one organism can base pair with the complimentary end of DNA,
segment from a second organism.
Another enzyme, known as ligase, can then covalently link the two molecules of DNA together.
By this procedure, DNA fragments (genes) from one organism can be inserted,
into the DNA of a second organism to form recombinant DNA.
When the recombinant DNA is inserted into a living cell,
its genetic message is transcribed into mRNA along with the messages from the host’s genes,
and then translated into protein.
Recombinant DNA has greatly increased our knowledge of genes and how they work.
It has led to the isolation of specific genes that can be transferred by recombinant DNA into bacteria,
so that the multiplication of the bacteria, will replicate the recombinant DNA, along with the host DNA,
producing large amounts of cloned DNA.
Determination of the nucleotide sequence of this cloned DNA,
can then be used to indirectly determine the amino acid sequence of the protein coded by the gene.
The amino acid sequences of many proteins are now being determined in this manner.
This technical procedure is easier than determining the amino acid sequence from a protein directly.
The potential benefits of this recombinant technique are many.
For example, inserting the gene that codes for human insulin into bacterial DNA,
leads to the production of insulin by the bacteria.
This hormone can then be extracted and used to treat diabetic patients,
who are unable to synthesis their own insulin.
In the future, it is hoped that the recombinant DNA technique,
may provide the ability to selectively replace in humans,
mutant genes that cause inborn errors in metabolism, with normal genes,
and thus provide a cure for these diseases.
On the other hand, the technology also bears potential hazards.
For example, it would be theoretically possible to insert a gene that codes for a toxic protein,
into a harmless bacteria, that commonly inhabits the human gastrointestinal tract,
and thereby produce a wide spread epidemic.
It is hoped that the benefits of genetic engineering,
will outweigh the hazards of being able to manipulate an organism’s genes.