Download GENE EXPRESSION - PROTEIN SYNTHESIS A. FROM DNA TO

GENE EXPRESSION - PROTEIN SYNTHESIS
A. FROM DNA TO PROTEIN: THE ROLE OF RNA
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
messenger RNA (mRNA) - single stranded; contains codons (3
.1
.base codes); mRNA is constructed to copy or transcribe DNA sequences
ribosomal RNA (ribosomes!) (rRNA) - ribosomes "read" the code
on the mRNA molecule & send for the tRNA molecule carrying the
.appropriate amino acid
.2
transfer RNA (tRNA) - clover leaf shaped; at least one kind for each
.3
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
RNA nucleotides contain a different sugar than DNA nucleotides.
.)(ribose vs. deoxyribose
.RNA is single stranded - DNA is double stranded
.1
.2
In RNA, uracil replaces thymine. There is no thyamine in RNA!!!
.But, there is adenine
.3
:B. TWO MAJOR EVENTS IN PROTEIN SYNTHESIS
]Transcription [mRNA copies or transcribes DNA sequences .1
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
Translation - actual synthesis of polypeptides or proteins; translate .2
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
.The genetic code. The mRNA codons for the 20 universal amino acids
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
V. MUTATIONS
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
Chemical Mutagens – ex. Nitrates and nitrites are added to foods .1
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
Physical Mutagens - Include UV light, X-rays, gamma radiation, &
.decay of radioactive elements; heat is slightly mutagenic
.2
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
VI.
GENETIC TRANSFER
Gene transfer refers to the movement of genetic information between
organisms. In most eukaryotes, it is an essential part of the organism’s
life cycle and usually occurs by sexual reproduction. Male and female
parents produce sperm and egg which fuse to form a zygote, the first
cell of a new individual. Of course, sexual reproduction does not occur
in bacteria, but even they have mechanisms of genetic transfer. Gene
transfer is significant because it greatly increases the genetic diversity of
organisms. We’ve already discussed how mutation account for some
genetic diversity, but gene transfer between organisms accounts for
even more. In recombinant DNA technology, genes from one species of
organism are introduced into the genetic material of another species of
organism. For example, human genes can be inserted into the bacterial
.chromosome
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
.Plasmids can move in and out of the bacterial chromosome
Two important plasmids are fertility (F) plasmids and drug
.resistant (R) plasmids
.2
.3
The F Plasmid - This plasmids contains about 25 genes, many of .1
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
The R Plasmid - In 1959 a group of Japanese scientists discovered .2
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,