Chap.
9 & 10 – Microbial Genetics (DNA Replication & Protein Synthesis),
Recombinant DNA,
In
the Watson-Crick model, the DNA molecule is a double-stranded helix, shaped like a twisted
"ladder." Remember that nucleic
acids (DNA & RNA) are made up of building blocks called nucleotides.
Each nucleotide is made up of a sugar, a phosphate, and a nitrogenous base. When we put these nucleotides together to build a
DNA ladder, the sides of the ladder are composed of alternating phosphate groups
& sugar molecules. The rungs
of the ladder are made up of paired nitrogenous bases joined in the middle by
hydrogen bonds. The nitrogenous bases are adenine, thymine, guanine, & cytosine; adenine always pairs with thymine (A-T
or T-A) & guanine always pairs with cytosine (G-C or C-G) [This is called complementary base pairing]. These 4 bases spell out the genetic message or
code!
DNA
enters into 2 kinds of reactions:
1.) Replication
- replicates the DNA before cell division, so that each new daughter cell will receive a
copy.
2.) Protein Synthesis (Gene Expression); 2 steps: transcription & translation
III. DNA REPLICATION IN PROKARYOTES
[Remember
bacteria have a circular chromosome.]
Replication
begins by an enzyme breaking the hydrogen bonds between the nitrogenous bases in the DNA
molecule; the double stranded DNA molecule "unzips" down the middle, with the
paired bases separating. As the 2 strands
separate, they act as templates, each one directing the synthesis of a new complementary
strand along its length.
If
a nucleotide with thymine is present on the old strand, only a nucleotide with adenine can
fit into place in the new strand; if a nucleotide with guanine is present on the old
strand, only a nucleotide with cytosine can fit into place in the new strand, & so on. This is called complementary base pairing. DNA replication is called semiconservative replication since
half of the original DNA molecule is conserved in each new DNA molecule. Like
other biochemical reactions, DNA replication requires a number of different enzymes, each
catalyzing a particular step in the process.
IV. GENE EXPRESSION - PROTEIN SYNTHESIS
By
the 1940's biologists realized that all biochemical activities of the cell depend on
specific enzymes; even the synthesis of enzymes depends on enzymes! Remember that the DNA molecule is a code that
contains instructions for biological function & structure. Proteins (enzymes) carry out these instructions. The linear sequence of amino acids in a protein
determines its 3-D structure & it is this 3-D structure that determines the protein's
function. The big question was: How does the sequence of bases in DNA specify the
sequence of amino acids in proteins? The
search for the answer to this question led to the discovery of RNA (ribonucleic acid),
which is similar in structure to DNA (deoxyribonucleic acid).
Three
types of RNA:
1. messenger
RNA
(mRNA) - single stranded; contains codons (3
base codes); mRNA is constructed to copy or transcribe DNA sequences.
2. ribosomal
RNA
(ribosomes!) (rRNA) - ribosomes "read" the code on the mRNA molecule & send
for the tRNA molecule carrying the appropriate amino acid.
3. transfer
RNA
(tRNA) - clover leaf shaped; at least one kind for each of the 20 a. a. found in proteins;
each tRNA molecule has 2 binding sites - one end, the anticodon (also a 3 base code), binds to the codon
on the mRNA molecule; the other end of the tRNA molecule binds to a specific amino acid;
each tRNA & its anticodon are specific for an a. a.!!
Differences
between RNA & DNA:
1. RNA
nucleotides contain a different sugar than DNA nucleotides. (ribose vs. deoxyribose).
2. RNA
is single stranded - DNA is double stranded.
3. In
RNA, uracil replaces thymine. There is no
thyamine in RNA!!! But, there is adenine.
B. TWO MAJOR EVENTS IN PROTEIN SYNTHESIS:
1. Transcription [mRNA copies or transcribes DNA sequences]
This
process is similar to what occurs in DNA replication.
A segment of DNA uncoils unzips. Free
RNA nucleotides, are then added one at a time to one end of the growing RNA chain. Cytosine in DNA dictates guanine in mRNA, guanine
in DNA dictates cytosine in mRNA, adenine in DNA dictates uracil in mRNA, thymine in DNA
dictates adenine in RNA. This complementary
base pairing is just like what occurs in DNA replication.
An enzyme catalyzes this process. After
transcription the mRNA goes out in search of a ribosome.
This mRNA molecule will now dictate the sequence of a. a. in a protein in the next
step called translation.
2. Translation
- actual synthesis of polypeptides or proteins; translate information from one language
(nucleic acid base code) into another language (amino acids); remember, the sequence of
amino acids (the protein's primary structure) determines what the protein's 3-D globular
structure is going to be & structure determines function.
a.
Initiation
- Begins when the ribosome attaches to the mRNA molecule, reading its first or START
codon. The first tRNA comes into place to
pair with the initiator codon of mRNA (it occupies the peptide site in the ribosome). The START codon is AUG, which specifies the amino
acid methionine. All newly synthesized
polypeptides have to start with methionine.
b. Elongation
- The second codon of the mRNA molecule is then read and a tRNA with an anticodon complementary
to the second mRNA codon attaches to the mRNA molecule; with its a. a. this second tRNA
molecule occupies the aminoacyl site of the ribosome.
When both the P & A sites are occupied, an enzyme forges a peptide bond between
the 2 a. a. & the first tRNA is released. The
first tRNA cannot be released until this peptide bond is formed, as it will take its a. a.
with it!! The second tRNA is then transferred
from the A site to the P site & a third tRNA is brought into the A site. The ribosome continues to move down the mRNA
molecule in this fashion, "reading" the codons on the mRNA molecule & adding
amino acids to the growing polypeptide chain.
c.
Termination
- Toward the end of the coding sequence on the mRNA molecule is a codon that serves as a
termination signal. There are no tRNA
anticodons to complementary base pair with this codon.
Translation stops and the polypeptide chain is freed from the ribosome. Enzymes in the cell then degrade the mRNA strand.
[In
eukaryotic cells, the polypeptide is taken up by the rough e.r. & is modified into a
3-D protein; the proteins are then packaged into transport vesicles (a piece of the e. r.
pinches off around the protein); these vesicles transport the proteins to the golgi
complex for further modification; the finished protein is pinched off in a piece of golgi
membrane (another vesicle) and is transported to the part of the cell where it is needed. In the prokaryotic cell, none of these organelles
exist, modification/processing of the polypeptide into a protein occurs in the cytoplasm.]
See
the table in your text of mRNA codons for the 20 amino acids. The 3-base codons are written to the left and the
abbreviations of the amino acids they correspond to are written to the right.
The
amino acid abbreviations in the table are: Ala
- alanine; Arg - arginine;
Asn
- apararagine; Asp - aspartamine; Cys - cysteine; Glu - glutamic acid; Gln - glutamine;
Gly
- glycine; His - histidine; Ile - isoleucine; Leu - leucine; Lys - lysine; Met -
methionine; Phe - phenylalanine; Pro - proline; Ser - serine; Thr - threonine; Trp -
tryptophan;
Tyr
- tyrosine; Val - valine.
The
code has been proven to be the same for all organisms from humans to bacteria - it's known
as the universal genetic code.
Notice
that most of the amino acids have more than one code (ex. Arg has 6 codes!). However, each code is specific for an amino acid
(ex. UUU only codes for the amino acid Phe).
Three
of the 64 codons do not specify amino acids. Instead
they indicate STOP or termination of the translation process (they say "This is the
end of the polypeptide.")
The
START codon is AUG, which specifies the amino acid methionine. All newly synthesized polypeptides have to
start with methionine. Since AUG is the only
codon for methionine, when it occurs in the middle of a message, it is ignored as a START
codon and is simply read as a methionine-specifying codon.
A. A
mutation is any chemical change in a cell's genotype (genes) that may or may not lead to
changes in a cell's phenotype (specific
characteristics displayed by the organism). Many
different kinds of changes can occur (a single base pair can be changed, a segment of DNA
can be removed, a segment can be moved to a different position, the order of a segment can
be reversed, etc.). Mutations account for
evolutionary changes in microorganisms and for alterations that produce different strains
within species. Mutations often make an
organism unable to synthesize one or more proteins.
The absence of a protein often leads to changes in the organism'’ structure or
in its ability to metabolize a particular substance.
B. Spontaneous
mutations
– occur by chance, usually during DNA replication.
Only about one cell in a hundred million (108) has a mutation in any
particular gene. Since full-grown cultures
contain about 109 cells per milliliter, each milliliter contains about 10 cells
with mutations in any particular gene. Because
the bacterial chromosome contains about 3,500 genes, each ml of culture contains about
35,000 mutations that weren't present when the culture started growing. Wow, when you think about it that’s a lot of
mutations in just one ml!
C. Induced
mutations
are caused by chemical, physical, or biological agents called mutagens.
1. Chemical
Mutagens –
ex. Nitrates and nitrites are added to foods such as hot dogs, sausage, and lunch meats
for antibacterial action. Unfortunately these
same compounds have been proved to cause similar mutations and cancer in lab animals
2. Physical
Mutagens
- Include UV light, X-rays, gamma radiation, & decay of radioactive elements; heat is
slightly mutagenic.
D. Consequences
of Mutations
- Most mutations do not change the cell's phenotype.
If the mutation changes the codon to another that encodes the same amino acid, the
protein remains the same. For example if the
DNA code is changed from AGA to AGG, the mRNA codon would change from UCU to UCC. Check your table!
The amino acid would not change. The
amino acid would stay serine. In this case
the genotype is altered, but the phenotype stays the same.
Having more than one codon for each amino acid allows for some mutations to occur,
without affecting an organism’s phenotype. A
mutation that changes a codon to one that encodes a different a. a. may alter the protein
only slightly if the new a. a. is similar to the original one. However, if a mutation changes an a. a. to a very
different one, there may be a drastic change in the structure of the protein, causing
major complications for the cell. For
example, if the structure of an enzyme called DNA polymerase was greatly altered, the cell
would not be able to replicate its DNA and thus would not be able to multiply.
E. Repair
of DNA Damage – Bacteria
& other organisms have enzymes that repair some mutations.
A. BACTERIAL
PLASMIDS & CONJUGATION
Most
bacteria carry additional DNA molecules known as plasmids:
1. Plasmids
are circular DNA molecules, much smaller than the bacterial chromosome.
2. Plasmids
can move in and out of the bacterial chromosome.
3. Two
important plasmids are fertility (F) plasmids and drug resistant (R) plasmids.
1. The
F Plasmid - This
plasmids contains about 25 genes, many of which control the production of F pili. F pili
are long, rod-shaped protein structures that extend from the surface of cells containing
the F plasmid. Cells that lack the F plasmid
are known as female (recipient) or F(-) cells. Cells that possess the F plasmid are known as
male (donor) or F(+) cells. F(+) cells attach themselves to F(-) cells by
their pili and transfer a copy of an F plasmid to the F(-) cells through a pilus. The once F(-) cells are now F(+) and will now
produce pili, because they now have the F plasmid that contains the plasmid genes that
code for these pili. This transfer of DNA
from one cell to another by cell-to-cell contact is known as conjugation and is a form of sexual recombination
because new genetic material is introduced into the cell.
This is as close to sex as bacteria get!
2. The
R Plasmid
- In 1959 a group of Japanese scientists discovered that resistance to certain antibiotics
and other antibacterial drugs can be transferred from one bacterial cell to another. It was subsequently found that genes conveying
drug resistance are often carried on plasmids. Over
the last few decades, R factors have proliferated to the point that some infections are
difficult to cure with antibiotics.
Note: Plasmids
are very important to scientists involved in recombinant DNA research. Genes of interest can be inserted into plasmids. The plasmids are introduced to bacteria and the
bacteria take them up by endocytosis. As the
bacteria reproduce themselves by mitosis, they replicate the plasmid during interphase and
pass it to their daughter cells. The plasmids
can then be isolated from all of these bacterial cells and the gene of interest can be
excised. In this way a large quantity of a
gene of interest can be produced. We'll talk
about this more later.
B. TRANSFORMATION
- A
genetic change in which DNA leaves one cell, exists for a time in the aqueous
extracellular environment, & then is taken into another cell where it may become
incorporated into the genome. Ex. Extracts from killed, encapsulated, virulent (disease causing) bacteria, when added to
living, harmless, unencapsulated bacteria, can convert the latter to the virulent type. By
endocytosis, the living, nonvirulent bacteria pick up the DNA from the dead, virulent
bacteria and incorporate the DNA into their own DNA.
The nonvirulent bacteria now have the genes that code for proteins that transform
them into virulent bacteria.
A
LITTLE ABOUT VIRUSES
Viruses
consist essentially of a molecule of nucleic acid (RNA or DNA) enclosed in a protein coat
called a capsid. No cytoplasm, ribosomes, or other organelles
are present. Viruses move from cell to cell,
utilizing the host cell's chromosomes, enzyme systems, and organelles to replicate the
viral nucleic acid and synthesize new capsid proteins.
They are obligate parasites in that they can't multiply outside the host
cell. Viruses that infect bacteria are called
bacteriophages.
A
virus will attach to a host cell and inject its nucleic acid into the host cell. The viral nucleic acid takes over the host cell's
genetic material and causes it to help the virus replicate the viral nucleic acid and to
produce its capsids. The new viruses are
assembled in the host cell (nucleic acids are inserted into the capsids) and the host cell
is lysed to release the new viruses to go and attack other cells. This cycle is known as the lytic cycle.
Viruses that infect bacterial cells are called bacteriophages.
C. Temperance
or Lysogeny –
This mechanism gives viruses the capacity to set up long-term relationships with their
host cell. Instead of going through the lytic
cycle, the virus nucleic acid remains integrated in the host cell’s chromosome. The virus may remain in this latent stage for long periods of time before
initiating a lytic cycle. The problem with
this type of cycle is that the integrated viral nucleic acid gets replicated along with
the host cell's chromosome during cell division & is passed to daughter cells (&
then they pass it to their daughter cells, & so on). Something
(ex. temperature change, stress) may later trigger these
latent viruses to go into the lytic cycle all at once, destroying all of the infected host
cells.
D. Transduction - Viruses can serve as vectors or carriers of genetic
information from one bacterium to another. During
the reproduction of the virus in the bacterium, fragments of bacterial DNA instead of viral nucleic acid may
become accidentally incorporated into a viral capsid.
Such "viruses" may be able to infect a new host cell, but they are not
able to complete a lytic cycle. The genes
they carry from a previous bacterial host may become incorporated into the chromosome of
the new bacterial host, possibly giving the bacterium new characteristics (ex. drug
resistance).
A.
PROCESS:
For
example a particular human gene can be removed from a human chromosome. Recombinant DNA is then constructed by inserting
that gene into a bacterial plasmid, which serves as a carrier. The recombinant DNA is then introduced host
bacterium, which takes up the plasmid. The
host bacterial cell then divides and its daughter cells divide, producing millions of
cells that all contain a copy of the human gene of interest. This process serves at least 2 purposes:
1.) Large
quantities of the human gene of interest are produced.
2.) The
bacteria can read the human gene of interest, producing the protein coded for on the gene
by protein synthesis. The genetic code is
universal! We can obtain large quantities of
a particular protein using bacteria.
a.
Vaccines
- Immunization
means deliberately introducing an antigen into the body that can provoke an immune
response & the production of memory cells. The
first injection elicits a primary immune response, which provokes the production of
antibodies & memory cells to provide long-lasting protection against disease. Many vaccines are made from killed pathogens
(called inactivated vaccines); pathogens
can be killed/inactivated by heat or chemicals (such as formaldehyde). Too much heat denatures proteins (changes their
shape). When the body comes into contact with
the real thing it won't have the right antibodies and memory cells & the person may
get the disease. If the pathogens aren’t
heated enough, some live pathogens may be injected in the vaccine! Attenuated
(weakened) pathogens are also used in vaccines; these have been cultured in abnormal
conditions so that they are no longer pathogenic. Sometimes
these organisms cause adverse side effects & can sometimes revert to their pathogenic
forms, causing the disease.
Using
"genetically engineered viruses:" Recently,
genetic engineering & recombinant DNA technology has allowed us to use bacteria to
produce the protein antigens found in the protein capsids of certain viruses (remember,
viruses don't have phospholipid cell membranes - they have proteins coats or capsids). Scientists determine the genetic code for these
proteins & insert the gene into the chromosome of bacterial cells. The bacteria
produce the proteins coded for on the inserted genes when they go through their regular
process of protein synthesis. These proteins
can then be injected as a vaccine (your body doesn't care if the proteins are in the real
viral capsid or if they were made by a bacterium; they are the same proteins & your
body's immune system will respond to these antigens in the same way). "Genetically engineered viruses" (ex.
hepatitis B, influenza, rabies) do not pose the same risks as inactivated and attenuated
viruses!