Protein Synthesis, Gene Expression,

Mutations, and DNA Technology

 PROTEIN SYNTHESIS AND GENE EXPRESSION
RNA versus DNA
RNA or ribonucleic acid is responsible for transferring the instructions from the DNA in
the nucleus to the ribosomes in the cytoplasm for protein synthesis. Like DNA, RNA is a
polynucleotide (nucleic acid).

The main differences between RNA and DNA are:

1. RNA is usually short and single-stranded, whereas DNA is usually much longer and
double stranded.
2. RNA has a 5-carbon sugar called ribose, whereas DNA has the 5-carbon sugar called
deoxyribose (the difference is one less oxygen atom on the number 2 carbon in DNA
compared to RNA).
3. RNA has the nitrogenous bases (nucleotides) cytosine, guanine, adenine, and uracil,
whereas DNA has cytosine, guanine, adenine, and thymine. In other words, RNA
contains the nitrogen base uracil in place of thymine.
4. RNA is located both inside and outside the nucleus of the cell, whereas DNA is restricted
to the nucleus in eukaryotic cells because of its large size.
5. RNA functions to facilitate the creation of polypeptides (which will become functional
proteins), whereas DNA functions as the storage molecule for the hereditary information

Three Major Classes of RNA

1. Messenger RNA (mRNA): takes a message from DNA to ribosomes. In eukaryotic cells,
DNA is in the nucleus, ribosomes in the cytoplasm.
 2. Ribosomal RNA (rRNA): rRNA along with other proteins make up the actual ribosome
in the cytoplasm. rRNA helps read mRNA during protein synthesis.
3. Transfer RNA (tRNA): tRNA brings amino acids to the ribosomes during protein
synthesis where they will be stitched together to make polypeptides.

Messenger RNA (mRNA)

• Takes the genetic information or “message” from the DNA to the protein making
machinery (the ribosomes) in the cytoplasm
• mRNA copies the information for a single gene out of the nucleus as a short, single-
stranded RNA transcript consisting of RNA nucleotides that are grouped in triplets (these
triplets are called codons)
• This process of copying genetic messages from DNA to mRNA is called
TRANSCRIPTION, and requires the enzyme RNA POLYMERASE
• In a cell, the genetic message is encoded in the order of nucleotides on the DNA, and will
get transcribed into a complimentary sequence on an mRNA molecule
• Remember that DNA uses the nucleotides that have C, G, A, and T as their nitrogenous
bases. RNA has the bases C, G, A, and U. The transcript is made by complementary
base pairings.
Question: If the message on the DNA was TAC GGT CAG TCA, what would the RNA
transcript be?

Ribosomal RNA (rRNA)

• Ribosomes are made of ribosomal RNA and proteins
• The two subunits of the ribosome – the large ribosomal subunit and the small ribosomal
subunit – are the location where proteins are made.
 • Ribosomes are the location where the translation of the mRNA transcript is made
into polypeptides (proteins).

Transfer RNA (tRNA)

• Transfer RNA molecules bring individual amino acids to the ribosome, one-by-one in
order to build a polypeptide.
• Each tRNA molecule has a slightly different shape and carries a different amino acid.
• A tRNA molecule has an attachment site for a specific amino acid on one end. On the
other end of the molecule there is an anticodon that is the complement to the codon on
an mRNA molecule
• An anticodon on a tRNA molecule is a three (nucleotide) base set that is a
complementary match to a three-base codon on the mRNA

Protein Synthesis/Gene Expression

Gene Expression requires 2 steps:
1. TRANSCRIPTION
• Occurs in the nucleus
• DNA nucleotides are transcribed (copied) letter by letter into RNA nucleotides
2. TRANSLATION
• Occurs in the cytoplasm
• The RNA transcript from transcription is used to direct the sequence of amino acids
to help build a functional protein
Transcription: From DNA to RNA
• In transcription, the enzyme that opens the DNA molecule up so that a mRNA transcript
can be created is RNA polymerase
• The location where RNA polymerase attaches to the DNA strand to start transcribing a
gene is called the promoter
o The promoter is a region of a special sequence of nucleotides that “promotes” the
process of transcription
• Once the RNA polymerase binds to the promoter, the RNA transcript will begin to form
an mRNA molecule
• RNA polymerase opens the DNA helix at the promoter, it’ll then read a template strand
from 3’ to 5’, creating an RNA strand that is built in a 5’ to 3’ direction via
complimentary base pairing (Recall: strands created must run anti-parallel to the template
strand)
• When the new mRNA strand forms, it has a different sequence of bases complimentary to
the DNA strand that the RNA polymerase copied it from. We can also call this “fresh”
mRNA strand primary mRNA
• At the end of the gene segment, the RNA polymerase releases from the DNA. In bacteria,
the mRNA transcript is useable at release, it is a functional mRNA transcript that can go
to the ribosome for translation. In eukaryotes, the transcript must undergo processing.

Processing of Primary mRNA

• There are sections of nitrogen bases within the gene transcript that do not code for amino
acids and are not part of the actual code for the protein. These regions called intervening
sequences or introns, need to be removed.
• Removal of the introns from the transcript is called RNA splicing and is completed by
spliceosomes that are made of RNA and proteins and are almost as big as a ribosome
• Once the introns are removed, mRNA is left with only the necessary sequences and codes
for the synthesis of polypeptides. These regions that are needed for the gene product are
called exons and are the portions that will be expressed.
• Exons form the mRNA molecules that will “exit” the nucleus
Amino Acids
• There are only 20 different kinds of amino acids. To create all the different kinds of
protein molecules, the amino acids are arranged in a specific order. It is the sequence
and length of amino acids that determines the kind of protein.
• The sequence of nitrogen bases on a DNA strand determines the sequence of amino acids
in a protein molecule
• It takes three nitrogen bases to “code” for one amino acid. A segment of these three bases
in DNA is called a triplet. The equivalent triplet of 3 nitrogen bases on a mRNA strand is
called a codon.
• Since there are four different nitrogen bases, there are 64 possible triplet combinations.
The arrangement of codons along the mRNA constitutes the genetic code.
• Peptide bonds connect the amino acids together
• Codon Tables: some codon tables are circular, but most are square tables that have the
first base on the left side of the table, the second base across the top, and the third base on
the right hand side.

• Side notes:
o Three of the codons on mRNA are for stop signals and do not code for amino
acids
 o One codon is a start or initiator signal (AUG – methionine codon) which does
code for an amino acid
o One amino acid is often coded for by more than one codon
o Each codon codes for only one amino acid

Question: State which amino acid each codon listed below codes for.
1. GAA
2. CAC
3. ACA
4. CUU
5. AUG
6. UAG

Translation: RNA to Protein Synthesis

• Once transcription is complete, the mRNA moves out of the nucleus to the small
ribosomal subunit in the cytoplasm.
• The ribosome is the site where translation occurs
• Translation is the process where the genetic code of messenger RNA (mRNA) is
deciphered to make proteins.
Translation in 3 Steps:
1. Chain Initiation – the steps necessary to begin the process of translation
o During chain initiation, a small ribosomal subunit, the mRNA, an initiator tRNA
bound to a methionine, and a large ribosomal subunit all come together to create
the initiation complex
o The AUG (start) codon of the mRNA strand must be exposed in the ribosome so
that the initiator tRNA can pair its anticodon (UAC) with the mRNA codon
2. Chain Elongation – the steps necessary to bring about polypeptide synthesis
o The ribosome has a binding site for mRNA as well as two binding sites for tRNA
o The P site = the polypeptide site; this is the site where the growing
polypeptide chain is forming
o The A site = the amino acid arrival site
o Once the initiator tRNA has bonded with the mRNA, it will move from the A site
of the ribosome to the P site giving room for another tRNA to enter the ribosome
o As another tRNA molecule enters the A site of the ribosome, a peptide bond will
form between its amino acid and the methionine amino acid that is attached to the
initiator tRNA
o When the amino acids are linked together, the first tRNA detaches and leaves the
ribosome, which has shifted over, displaying the next codon, and making room
for the new tRNA molecule
3. Chain Termination – the steps necessary to end the process of translation
o This process continues until a stop codon is reached, at which the two ribosomal
subunits dissociate and fall off of the mRNA molecule

GENES AND GENE MUTATIONS

A mutation is any heritable change in the sequence of DNA, including:
• Chromosomal mutations: a change in the total number of chromosomes or in the structure
of a chromosome
• Gene mutations: involves one or more changes in the nitrogenous base sequence of a
gene
When proteins are being synthesized, mutations can occur. These mutations can be caused by:
• Replication errors (also known as spontaneous mutations) = a rare source of mutations as
DNA polymerase proofreads the new mRNA strand vs. the old strand. If there are
mismatched pairs then they are replaced with the correct nucleotides.
• Mutagens (also known as induced mutations) = chemical or physical agents from the
environment. Mutagens might include radiation (ex. radioactive elements, X-rays,
ultraviolet (UV) radiation) and certain organic chemicals (ex. chemicals in cigarette
smoke and certain pesticides)
• Transposons = are specific DNA sequences that have the remarkable ability to move
within and between chromosomes. Their movement to a new location sometimes alters
neighboring genes, particularly by increasing or decreasing their expression.

The result of these mutations can be:

1. Harmful: where there is usually a loss of function
2. Helpful: usually a gain of function; but some gain of function can benefit cancer cells, so
it’s helpful to the cancer cells but harmful to the organism
3. Silent or Neutral: no noticeable effect on the organism

Types of Mutations
Two types of mutations are: frameshift mutations and point mutations.

Frameshift Mutations
• A frameshift refers to the “reading frame” of the sequence.
• This type of mutation takes place where there is an addition or deletion of one or more
nucleotide bases.
• Everything after the point of insertion/deletion is changed, so a frameshift mutation
“shift” the reading frame of codons in the mRNA.
• Furthermore, frameshift mutations are usually more severe than point mutations since
they affect every amino acid whose codon is after the mutation.
• Frameshift mutations can be extremely damaging.
Point Mutation
• A point mutation is a change in a single nucleotide base in a gene.
• A single nucleotide change may not change the amino acid that is brought into the
polypeptide.
• Consequently, a point mutation may or may not result in a change in the amino acid
sequence of the encoded protein.

Point Mutations
Changes at a single point in the DNA Sequence

Silent Nonsense Missense

Mutation causes no A mutation that results Results in a change in the
change in the amino acid in a premature stop amino acid sequence
sequence in the end. codon

• A substitution error that still codes for the same amino acid is called a silent mutation
because it has no noticeable effect on the polypeptide formed.
For example, glycine’s codons include GGU, GGC, GGA, and GGG. If the DNA
sequence was CCA and a point mutation occurred on the mRNA transcript
making it GGC instead of GGU, then glycerine would still be the amino acid in
the chain.
 • A substitution error that changes one amino acid for another is called a missense
mutation. The amino acid is altered, but if it is at only one point, it has little effect on the
protein as a whole.
If the error was in the first base and it was AGU instead of GGU, the amino acid
would be serine instead of glycine. This may not be a problem in a very large
protein, but if the error was in the active site of an enzyme, it could affect its
function.
• A substitution, deletion, or addition of three successive nucleotides would have the same
missense effect. They could change the amino acid at that one location only.
• If the new bases cause a stop codon to be created (called a nonsense mutation) that
would be a problem, especially if it occurred early in a sequence. No part of the
polypeptide after the stop would be created.

DNA TECHNOLOGY
The process of manipulating the genes of organisms, such as cloning the genes and using them to
alter the genomes of viruses and cells (whether they be bacteria, plant, or animal cells) is called
genetic engineering.

The Cloning of a Gene

• Gene cloning is the production of many identical copies of the same gene.
• Biologists clone genes for a number of reasons:
o To determine the difference in base sequence between a normal gene and a
mutated gene
o Might use the genes to alter the phenotype of other organisms in a beneficial way
§ In humans, this is called gene therapy
§ In other organisms, we call this a transgenic organism
• Begin by using one of the two ways to clone the gene:
1. Using Recombinant DNA Technology
2. Using the Polymerase Chain Reaction (PCR)
Using Recombinant DNA Technology
• Recombinant DNA (rDNA) contains DNA from two or more different sources
• To make rDNA, a researcher needs a vector (which is a piece of DNA that can be
manipulated) in order to add foreign DNA to it
• Plasmids are small accessory rings of DNA from bacteria that are not part of the
bacterial chromosome and are capable of replicating on their own (plasmids are one of
the more common vectors used when trying to make rDNA).
• In order to introduce foreign DNA into vector DNA, two different enzymes are required:
1. A restriction enzyme to cleave DNA
2. DNA ligase to seal DNA into an opening created by the restriction enzyme
• Restriction Enzymes
o Called this because they restrict the growth of viruses.
o Can also be used as molecular scissors to cut double-stranded DNA at a specific
site.
o In order to ensure that the vector DNA and foreign DNA have complimentary
base pairs, scientists will cleave the foreign DNA with the same restriction
enzyme that was used to cleave the vector DNA.
• The single-stranded, but complementary, ends of the two DNA molecules are called
“sticky ends” because they facilitate the insertion of foreign DNA into vector DNA.
• DNA Ligase
o An enzyme that functions in DNA replication to seal any breaks in the double-
stranded helix, is then used to seal the foreign piece of DNA into the vector.

Using the Polymerase Chain Reaction (PCR)

• The Polymerase Chain Reaction (PCR) requires the use of a DNA polymerase, a set of
primers, and a supply of nucleotides for the new DNA strands.
• PCR creates millions of copies of a segment of DNA very quickly without the use of a
vector or a host cell.
• PCR is a chain reaction because the targeted DNA is repeatedly replicated as long as the
process continues
 • Automated PCR machines became possible after the enzyme Taq polymerase was
extracted from the bacterium Thermus aquaticus
• During the PCR cycle, the mixture of DNA and primers is heated to 95ºC to separate the
two strands of the DNA so that the primers can bind to single-stranded DNA
• Taq polymerase is used to separate double-stranded DNA as it can withstand the high
temperature that the DNA is being put through

DNA Fingerprinting
• DNA Fingerprinting is based on the differences in the sequence of DNA nucleotides
between individuals.
• In order to measure DNA fragment length, a technique called gel electrophoresis is used
• During gel electrophoresis, DNA fragments migrate through a jellylike material (the gel)
according to their length when an electrical field is applied.
o The shorter the fragment, the farther it migrates
• DNA fingerprinting has many uses including:
o Medically, it is used to identify the presence of a viral infection or a mutated gene
that could predispose someone to cancer
o In forensics, DNA fingerprinting from a single sperm is enough to identify a
suspected rapist (possible thanks to PCR)
o Can identify the parents of a child or identify the remains of someone who died

Biotechnology
• Biotechnology uses natural biological systems to create a product or to achieve an end
desired by human beings.
• Organisms that have had a foreign gene inserted into them are called transgenic
organisms

Transgenic Bacteria
• Recombinant DNA technology is used to produce transgenic bacteria
• Biotechnology products produced by bacteria include:
o Insulin
 o Human growth hormone
o t-PA (tissue plasminogen activator)
o Hepatitis B vaccine
• There are many non-human uses for transgenic bacteria as well
o Promote the health of plants
o Insect toxin (protect the roots of plants from insects)
o Degrade substances (ex. genetically engineered to clean up beaches after oil
spills)

Transgenic Plants
• Foreign genes have been transferred to crops such as cotton, corn, and potato to make
them resistant to pests (cells now produce insect toxin)
• Soybeans have been made resistant to a common herbicide
• Hope is that plants can one day be engineered to have leaves boost their CO2 intake or cut
down on water loss
• Plant pharming is the use of engineered plants to produce human proteins, such as
hormones, clotting factors, and antibodies, in their seeds

Transgenic Animals
• Techniques have been developed to insert genes into eggs of animals
• Using this technique, many types of animal eggs have acquired the gene for bovine
growth hormone (bGH)
• The procedure has been used to produce larger fish, cows, pigs, rabbits, and sheep
• Animal pharming, the use of transgenic farm animals to produce pharmaceuticals, is
being pursued by a number of firms
• Plans are underway to produce drugs for the treatment of cystic fibrosis, cancer, blood
diseases, and other disorders by this method

Control of Gene Expression

Totipotent = a cell has the complete potential (contains all the genes necessary) to develop into
an organism
Reproductive Cloning
• In reproductive cloning, the desired end is an individual that is genetically identical to
the original individual
• Cloning of plants has been done for a while, but it was thought that cloning animals
would be impossible
• Dolly the sheep
o Dolly the sheep was the first ever animal to be cloned back in 1997
o Now, it is common practice to clone all sorts of farm animals that have desirable
traits and even to clone rare animals that might go extinct
o Dolly, however, did not live a very healthy life as she only lived half the normal
life span for a Dorset sheep after suffering from lung cancer and arthritis

Therapeutic Cloning
• In therapeutic cloning, the desired end is not an individual, but rather it is mature cells
of various cell types
• The purpose of therapeutic cloning is:
1. To learn more about how specialization of cells occurs
2. To provide cells and tissues that could be used to treat human illnesses, such as
diabetes, spinal cord injuries, Parkinson disease, and so on
• Two possible ways to carry out therapeutic cloning:
1. Embryonic stem cells are used, separated, and are each subject to a treatment that
will cause them to develop into a particular cell type
2. Use of adult stem cells to hopefully become any type of specialized cell

Gene Expression in Bacteria – Lac Operon

• Three enzymes are needed for bacteria to properly digest lactose
• The following DNA components are important for this to take place and they are:
o Promoter Gene: the binding site for an enzyme, RNA polymerase, that starts
transcription.
o Structural Gene: is a DNA segment that produces an mRNA by transcription
 o Operator Gene: situated next to the structural genes; when activated, it causes
transcription to occur in the structural genes.
o Regulator Gene: located outside the operon; responsible for the production of the
lac repressor; this protein binds to the operator gene in the absence of the
substrate, lactose
• Absence of Lactose in Environment
o Lac repressor binds to an operator gene, preventing RNA polymerase from
attaching to the promotor
o Because of this, transcription cannot occur and mRNAs for lactose-digesting
enzymes will not be produced
o Regulatory gene is located outside of the operon and codes for the repressor
making sure that the operon stays off until lactose is present
• Lactose Present in Environment
o Lac repressor binds with lactose, preventing the repressor from attaching to the
operator gene
o This allows RNA polymerase to attach to the promoter and signals the operator to
start transcription of the structural genes
o Production of genes needed for lactose metabolism occurs

Gene Expression in Eukaryotes

Levels of Gene Control
• Unpacking of DNA
o Some inactive genes are located within darkly stained portions of chromatin,
called heterochromatin
o Active genes in eukaryotic cells are associated with more loosely packed
chromatin, called euchromatin
o In order for transcription to begin, a chromatin remodeling complex needs to push
aside the histone portion of the nucleosome
• Transcription (Transcriptional)
 o Transcriptional control, just like in prokaryotes, is dependent on the interaction of
DNA sequences (enhancers and promoters) with proteins (transcription factors
and activators)
o Transcription activator binds to an enhancer, that can be quite a distance from the
promoter (DNA must bend for this to occur)
o Transcription factors help RNA polymerase bind to a promoter in order to then
initiate transcription
• mRNA Processing (Post-Transcriptional)
o Introns, segments of mRNA that do not code for amino acids in the polypeptide,
are removed
o Exons, segments of mRNA that do code for amino acids, are spliced together
o An altered Guanine nucleotide cap and a poly-A tail are added
o Nuclear pore determines the time it takes for an mRNA to enter the cytoplasm
• Translation (Translational)
o The longer an mRNA remains in the cytoplasm before it is broken down, the
more gene product there will be and therefore, the greater the amount of protein
formed
o mRNA is degraded by ribonuclease
o Availability of amino acids, tRNA, and subunits of rRNA determines the
translational rate
• Protein Activity (Post-Translational)
o Polypeptide takes a specific shape
o Activity of many proteins is short-lived before they are degraded or destroyed by
proteasomes