UNIT 8 NOTES – MOLECULAR BIOLOGY AND EMBRYONIC DEVELOPMENT
FROM GENES TO PROTEINS
I.
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Overview
Although all cells with a nucleus in our body contains the same genetic information but the
same set of genes may be expressed very differently at different stages in an organism’s life or
in different cells.
Mendel discovered that traits are passed from one generation to the next or could skip a
generation.
Hershey and Chase discovered that DNA is responsible for passing the genetic information on
from one generation to the next.
Archibald Garrod – as he studied alkaptonuria, he observed that some diseases are caused by
defective enzymes that stopped metabolic pathways.
Beadle and Tatum – used bread mold to cause mutations in them. Came up with the “One gene
– one enzyme hypothesis” with a set of experiments that followed the procedure below:
As more information was discovered, this hypothesis was modified to one gene only determines
one polypeptide, not necessarily a fully functioning protein.
II.
The Central Dogma of Biology
 Francis Crick created the term. According to the central dogma information is always inherited
from the DNA to the RNA to the proteins.
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DNA is considered a permanent molecule in the cell’s life while RNA is temporary and breaks
down quickly after it is used.
Different types of cells make different proteins depending on their functions. So different genes
are active in them.
Review the structure of RNA and its types (mRNA, tRNA, rRNA and siRNA)
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III.
Transcription is the process that converts the information from the DNA into RNA.
Translation converts the information from RNA to proteins.
Transcription
A. Molecular Components:
 An enzyme called RNA polymerase opens the two strands of the DNA molecule and hooks
together the RNA nucleotides as they base-pair along the DNA. RNA polymerase can only
assemble the polynucleotide chain from the 5’ → 3’ direction but they don’t need priming to
start the assembling. ONLY THE 3’
5’ DNA TEMPLATE IS COPIED.
 There are specific regions on the DNA where the assembling of the new mRNA molecule starts.
The sequence where RNA polymerase attaches and initiates transcription is the promoter. In
prokaryotes, the sequence that ends transcription is called the terminator. The promoter
region is said to be “upstream” from the terminator region. The stretch of DNA that is being
transcribed into an mRNA molecule is called the transcription unit.
B. Synthesis of an RNA Transcript:
 The three stages of transcription are: initiation, elongation and termination of the RNA chain.
 Initiation: It starts at the promoter region of a gene. This region includes the actual start point
of transcription and several dozen other nucleotides “upstream”. The promoter region binds
the RNA polymerase and determines which DNA strand will be copied. In prokaryotes the RNA
polymerase directly recognizes the promoter region and binds to it. In eukaryotes, a collection
of proteins called transcription factors mediate the binding and initiates transcription. The
transcription factors, RNA polymerase and the promoter region together are called
transcription initiation complex. The specific region of the promoter that has a set of repeating
TATA bases is called the TATA box.
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IV.
Elongation: RNA polymerase moves along the DNA molecule, it continues to untwist the DNA 10
– 20 nucleotides at a time and adds nucleotides to the 3’ end of the RNA molecule. The
nucleotides are used in a form of ATP, GTP, UTP and CTP, so energy is provided by them to form
the new bonds of the forming RNA molecule. Several polymerase molecules can transcribe the
same gene at the same time. Once the RNA molecule is ready, it peels off of the DNA molecule
and the DNA twists back again.
Termination: This mechanism is different in prokaryotes and eukaryotes. In prokaryotes, the
process of transcription continues through the terminator sequence of DNA. This sequence
makes the RNA polymerase detach from the DNA molecule and release the transcript which is
completely done. In eukaryotes, the pre-mRNA is made by the RNA polymerase but there is a
long additional sequence of polyadenilation signal (AAUAAA) and other additional nucleotides
“downstream” from the original gene that was copied. These additional nucleotides make the
proteins that were associated with the transcription fall off and the pre-mRNA released.
However, the pre-mRNA has to go through an editing process before it can be used as a
functional mRNA molecule.
RNA modification
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Alteration of mRNA ends: The 5’ end that is transcribed first gets a modified guanine nucleotide
forming a 5’ cap. The 3’ end gets additional 50 – 250 adenine nucleotides forming a poly-A tail.
The 5’ cap and the poly-A tail seem to facilitate the export of the mature mRNA from the
nucleus. They also help to protect the mRNA molecule from degradation by hydrolytic enzymes.
They also help ribosomes to attach to the 5’ end of the mRNA molecule.
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RNA splicing: the removal of a large portion of the RNA molecule by a “cut-and-paste” method.
The removed noncoding sequences of mRNA nucleotides that lie between the coding regions
are called introns. Exons are the coding sequences. The signal for cutting is a set of small
sequences of nucleotides that are recognized by small nuclear ribonucleoproteins (snRNP’s or
“snurps”). These snurps join with other proteins to form a spliceosome – molecular units that
cut introns out and attach exons to each other.
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The consequence of DNA splicing is that a single gene can encode more than one kind of
polypeptide. Depending on which segment of the gene is treated as an exon, it can give rise to
multiple polypeptide – alternative RNA splicing.
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Great, detailed movie on transcription: http://vcell.ndsu.nodak.edu/animations/transcription/movie.htm
Same site on RNA processing: http://vcell.ndsu.nodak.edu/animations/mrnaprocessing/movie.htm
http://highered.mcgrawhill.com/sites/0072507470/student_view0/chapter3/animation__mrna_synthesis__transcription___quiz_
1_.html
RNA splicing: http://www.sumanasinc.com/webcontent/animations/content/mRNAsplicing.html
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V.
The Genetic Code
 Translation is the process of converting information from mRNA into proteins.
 The information on the mRNA molecule is read in 3 nucleotide sequences called codon.
 All codons and what amino acids they determine is included in a table of codons. This table also
includes 1 start codon (AUG) that codes for methionine amino acid and starts the synthesis of
every polynucleotide chain. 3 stop codons are also included on the table. If these codons are
reached, the translation process will stop even if there is some more mRNA remains.
 The genetic code is nearly universal to all known species on the planet – another evidence of the
uniform origin of life.
 Once the mRNA code is being read, every nucleotide is only part of one codon and the reading
of codons is continuous, no leftover nucleotides between codons.
VI.
Translation
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Transfer RNA (tRNA): Its function is to transfer amino acids from the cytoplasmic pool of amino
acids to a ribosome. The tRNA molecules are not identical. Each tRNA molecule attaches to a
specific mRNA codon and carries a specific amino acid that matches that codon. One end of the
tRNA molecule is attached to a specific amino acid while the other end has a nucleotide triplet
called anticodon that is complementary to the codon of the mRNA molecule.
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Ribosomes – Small organelles that attach the proper tRNA molecules with the mRNA codons.
Ribosomes are made up of two subunits that are made in the nucleus of eukaryotes from
proteins and ribosomal RNA (rRNA). The large and small subunits bind only when they are
attached to an mRNA molecule. Prokaryotic ribosomes are smaller and lighter than eukaryotic
ones. Each ribosome has a binding site for the mRNA molecule and three binding sites for tRNA:
P site (holds the tRNA carrying the growing polypeptide chain), A site (holds the tRNA carrying
the next amino acid to be added to the polypeptide chain) and E site (the site that releases the
tRNA molecule.
A. Building a Polypeptide:
 Ribosome association and initiation: This stage brings together mRNA, a tRNA bearing the first
amino acid and the two subunits of a ribosome. First the small subunit binds with the mRNA
and the tRNA molecule that brings methionine. The small subunit than moves downstream
along the mRNA until it reaches the start codon. Once the tRNA binds to the initiation codon
with hydrogen bonds the large subunit also attaches to the small subunit. Proteins called
initiation factors are also important for this process. Energy is provided by GTP molecules.
 During elongation amino acids are added one at a time. Each addition involves the participation
of several proteins called elongation factors.
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Elongation continues until a stop codon on the mRNA molecule is reached on the A site of the
ribosome. A protein called a release factor binds to the stop codon and causes the addition of a
water molecule instead of another amino acid to the polypeptide chain.
A single mRNA molecule can be used to make many copies of a polypeptide simultaneously
because several ribosomes can work on the same mRNA molecule. These strings of ribosomes
are called polyribosomes or polysomes.
B. Modifications of the polypeptide chain:
 During the translation, the polypeptide chain begins to fold into its 3D shape. Additional steps
may be required such as adding carbohydrates (glycosylation), lipids, phosphate groups
(phosphorylation) to some amino acids.
 One polypeptide chain can be cut into multiple pieces or more than one polypeptide chains can
come together to form a functional protein.
 Some proteins are made in a form of an inactive precursor and activated only if some parts of it
are cleaved off – used in some enzyme activations.
 There are two types of ribosomes available. One type is free moving in the cytosol, the other is
attached to the ER or to the nuclear envelope. Free ribosomes synthesize proteins that are free
moving in the cytosol. Membrane-bound ribosomes make proteins of the endomembrane
system and proteins that are released for secretion. However, both types start out as free
ribosomes and attach the rough ER only during the translation process when certain signal
molecules make it.
 Western Blot – is a technique that is used to identify particular proteins in a cell or tissue
sample. Steps of this method:
o First proteins are separated by using gel electrophoresis by size.
o Next, the gel with the separated proteins is transferred to a blotting paper. The paper
absorbs the proteins in the same pattern as they were in the gel. This is the blot
o The paper than blocked by inactive proteins
o To look for one specific protein of interest on the blot, an antibody is added that will
only bind to the specific protein. Than a secondary antibody is added that binds to the
primary antibody. This secondary antibody will bind to a dye molecule or some other
substrate that makes the protein visible on the blot paper.
Animation: http://www.dnatube.com/video/1511/Western-blot
PROKARYOTIC GENE EXPRESSION
As a related topic please review bacterial genetic recombination including transformation, transduction
and conjugation
I.
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Gene Regulation in Bacteria
In bacteria transcription and translation are not separated by a barrier. In fact, these two
processes can take place at the same time. As soon as part of an mRNA is copied it can be
translated into a protein.
Also, RNA modification does not occur in bacteria
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Bacterial genes are organized into operons – a sets of genes that are transcribed together under
one regulatory unit of an operator and a promoter. These genes determine proteins that are
part of the same metabolic pathway.
 Promoter – is the region of the DNA that RNA polymerase binds with and transcription factors
also attach to it to start transcription.
 Operator – a specific sequence within the DNA that binds transcription factors to turn
transcription on or off. It is found within the promoter or between the promoter and the coding
region.
 An operon = promoter + operator + coding genes
II.
Repressible operons
 Repressible operons usually have their transcription on and they are repressed by a small
molecule that binds allosterically to a regulatory protein.
 Repressible operons produce important molecules such as certain amino acids that are
necessary to build macromolecules. If the final product (such as an amino acid) is available in
the environment, the bacterium does not need to produce it so it shuts down (represses) the
operon that results in the important molecule.
 When the operon is working (not repressed), the RNA polymerase can bind to the promoter
region, slide along the operator and copy the five genes for five different polypeptides to make
the enzymes of the tryptophan making metabolic pathway.
 During the working condition of the operon, there is an inactive repressor protein in the cell.
This repressor protein cannot bind alone to the operator so it just remains inactive.
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When tryptophan is available in the environment, the bacterium takes it in and uses it. In this
case the bacterium does not need to make tryptophan. One of the tryptophan molecules binds
with the repressor protein and activates it. This active repressor binds to the operator and acts
as a roadblock to the RNA polymerase. The polymerase cannot transcribe the gene so mRNA is
not produced.
http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120080/bio26.swf::The
%20Tryptophan%20Repressor
III.
Inducible Operons
 These operons are usually turned off and the genes are not being actively transcribed.
However, these genes can become active when an allosteric activator (effector) is introduced
into the cell.
 For example, bacteria usually don’t use lactose as a source of sugar, so they don’t have enzymes
to digest it. However, if lactose is introduced in the environment, they can activate genes to
produce lactose digesting enzymes.
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So when the operon is turned off, a repressor protein is attached to the operator and does not
allow transcription of RNA molecules.
When the bacterium finds lactose in the environment and eats it, one of the lactose molecules
can bind with the repressor and detach it from the operator. As the roadblock is removed, the
RNA polymerase can move along the gene and transcribe RNA molecules to make enzymes to
digest lactose.
Once the lactose is used up the repressor becomes active again and attaches back to the
operator and stops transcription.
https://highered.mcgraw-hill.com/sites/dl/free/0072835125/126997/animation27.html
EUKARYOTIC GENE REGULATION
I.
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Mechanisms of Gene Regulation in Eukaryotic Cells
Cell differentiation – multicellular organisms have very different cells with a wide range of
shapes and functions. They all contain the same DNA but different genes are turned on or off in
each. The process of cell differentiation turns some genes on and others off.
While prokaryotic gene regulation is mostly determined by environmental factors and involve
activators, repressors and operons; eukaryotic gene regulation can occur in different stages and
can occur for many different reasons.
The following table is a great review of the different stages at which gene regulation can take
place:
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II.
Regulation of Chromatin Structure:
Heterochromatin -- tightly packed parts of chromatin that stains dark under the microscope. Genes
here are usually not expressed.
Euchromatin -- part of the chromatin that is more loose and usually holds actively transcribing genes
Histone acetylation -- acetyl groups can be attached to histone proteins to prevent the histones to
bind with each other and tightly pack up. As a result, the chromatin remains lose and easily
transcribed.
Histone methylation -- methyl groups (-CH3) are attached to the histone tails. This promotes the
condensation (close packing) of the chromatin and makes it less active.
DNA methylation -- enzymes can also methylate certain bases of the DNA molecule. The
methylated sections of DNA are usually not transcribed. This can lead to long-term inactivation or
genomic imprinting -- the inactivation of the mother's or father's genes in diploid cells at the start of
development.
Epigenetic inheritance -- the modification of the chromatin can be inherited although these
modifications do not change the nucleotide sequence of the DNA. These modifications however,
can also be reversed. Epigenetic inheritance is changes in the phenotypic expression of a trait, but
this change is not due to changes in the DNA sequence. Other examples: all of what comes in these
notes, Barr body deactivation, lac operon in bacteria, Trp operon in bacteria.
III.
Regulation of Transcription Initiation:
 In a eukaryotic genes can be turned on or off during the initiation of transcription. The
complete initiation complex (several transcription factors and RNA polymerase II) have to be
assembled on the promoter region of the gene to initiate transcription. If only a few general
transcription factors bind to the TATA box, only a few mRNA molecules will be produced. To
have high efficiency translation performed, several specific transcription factors need to be
attached to the promoter region.
 The enhancer region can also improve the efficiency of transcription if activator proteins bind to
the enhancer region and those make the enhancer region bind to proteins of the transcription
initiation complex. As a result, the transcription can occur faster, more efficiently.
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IV.
Some specific transcription factors can also function as repressors to inhibit expression of a
particular gene. These repressors can prevent the binding of activators or other transcription
factors to the promoter region.
Mechanisms of Post-Transcriptional Regulation
Transcription alone will not result in gene expression. To have a functional protein, many things need to
take place after the pre-mRNA is made. Any one of the steps of making a functional mRNA or a
functional protein can be shut down or sped up as part of gene regulation. The following are a few
examples of these regulatory processes:
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RNA processing – Alternative RNA splicing is a good example of a way to form different mRNA
molecules.
mRNA degradation – mRNA has a limited life span to form proteins. Prokaryotic mRNA can be
broken down within minutes after transcription. In eukaryotes, however, mRNA can survive
longer, sometimes for weeks. This degradation of mRNA can be done by enzymes gradually
breaking down the 5’ cap, poly-A tail than the entire segment of mRNA. Small segments of RNA
molecules (microRNA’s or miRNA) can also attach to the mRNA and prevent translation or can
actually break down the mRNA.
Initiation of translation – regulatory proteins can bind to mRNA and prevent it to bind with the
ribosome during the initiation of translation
Protein processing and degradation – To activate proteins, they need to be modified by either
cleavage of certain parts off (pepsinogen to pepsin) or by attaching phosphate groups to the
protein (protein kinase receptors). Regulatory proteins can stop any of these steps and can
make proteins dysfunctional.
Quiz 1 is on the notes up to this point.