14.

1 Genes specify proteins via transcription and translation:

Evidence from the Study of Metabolic defects:

Early study of the expression of genes found that the lack or presence of enzymes was what caused

certain defects. Their presence is determined genetically, and also, the enzymes are involved in metabolic pathways

to actually express a phenotype.

Nutritional Mutants in Neurospora:

In this experiment, neurospora (a mold) was chosen because it is haploid. Researchers at Stanford

discovered that inactivating genes affected the phenotype of the organism.

They bombarded the cells with x-rays and tested to see how it affected their nutrition. They developed

various strains that could not metabolize some of the amino acids of the medium growth (Agar) that was given.

From further experimentation, they came up with the one gene- one enzyme hypothesis which explains that

enzymes are produced because specific genes code for them .

The Products of Gene Expression - A developing story:

The hypothesis was later modified since not all proteins are enzymes, and genes code for all proteins. But,

some proteins are a complex of polypeptide chains that can each come from genes. As a result, it was finally revised

to the one gene - one polypeptide hypothesis.

This isn’t always correct either since the genes first code for RNA, and sometimes the RNA does not go on

to become a polypeptide or a protein.

Basic Principles of transcription and translation:

DNA and RNA are made up of nitrogenous bases that can be thought of as ‘letters’. Polypeptides are

similar, however their letters are amino acids. Genes do not directly code for proteins, but instead use RNa to

translate between the two languages in two processes: transcription and translation.
 Transcription is the synthesis of a single strand of RNA by pairing with the strands of DNA. DNA acts as a

template for the RNA, and the only thing that is converted is any pair that would normally code for T instead codes

for U. The RNA that have the template of DNA impressed on them are known as Messenger RNA (mRNA).

Translation is the translation of the sequence of mRNA into amino acids that make up the polypeptide. This

occurs in ribosomes in the cytoplasm.

Transcription and Translation are the same in Eukaryotes and Bacteria. It is a little different in archaea,

however. In bacteria, since there is no nucleus, the translation can happen while transcription happens. In

eukaryotes there are other stages that occur within the nucleus.

14.2 Transcription is the DNA-directed synthesis of RNA - A closer look

Molecular components of Transcription:
First, the enzyme RNA polymerase separates the template and nontemplate DNA strands from each other. Then,

it starts adding RNA base pairs that are complementary to the template strand in the 5’ → 3’ direction. The segment

of DNA that is on the template strand and is involved in all of this is known as the transcription unit.

The RNA polymerase does not need a primer like the DNA polymerase.

A gene in a DNA template strand will however have a promoter which is a sequence that signals where the RNA

polymerase should start. In bacteria, there is a terminator as well, but there is a different mechanism for termination

in Eukaryotic cells.

Another difference in the Eukaryotic cells and bacteria cells for transcription is the types of RNA polymerase

that are used. For bacteria, there is only one polymerase that produces the different types of RNA. For Eukaryotes,

there are more than one, for example, RNA polymerase II is what produces mRNA.

Synthesis of an RNA Transcript:

RNA polymerase binding and initiation of transcription:

 Upstream in a strand means ‘to the left’ and Downstream means ‘to the right’ in a DNA strand. In other words,

upstream is in the 5’ → 3’ direction and vice versa.

In Eukaryotic cells, there are transcription factors along with RNA polymerase that help it bind to the

promoter. In bacteria, these extra molecules are not necessary. The entire complex with the factors and RNA

polymerase II is known as the transcription initiation complex.

The promoter is a series of nucleotides that end in the start point. This is where the actual RNA transcription

begins. The first step is initiation where the RNA polymerase binds and the DNA unwinds.

The crucial initiation sequences generally lie on the strand that is not the template strand. Transcription

factors recognize the TATA box and bind to it, other transcription factors attack, and then the polymerase binds.

Elongation of the RNA Strand:

Elongation is where the polymerase moves downstream and forms complementary base pairs that elongate the

RNA strand. As this is happening, the RNA strand is also pulling away and the DNA strand is being put back together

as well. Several polymerases can work on different genes at a time which speeds up protein synthesis.

Termination of Transcription:

Termination is where the polymerase detaches from the DNA, and the RNA transcription is complete.

In bacteria, there is a terminator signal as mentioned before.

In eukaryotes, there is a similar polyadenylation signal which the polymerase recognizes. After a few pairs are

added downstream, it detaches.

This is now the pre-mRNA which will go through further processing in the Eukaryotic nucleus.
 14.3 Eukaryotic cells modify RNA after transcription:
In RNA processing both ends are made shorter, and certain sequences in between are refined.

Alteration of mRNA Ends:

We know from before that the RNA is synthesized from 5’ → 3’. It has a 5’ cap that is made up of a modified

form of Guanine. At the end, the polyadenylation signal adds the AAUAAA sequence. This is at the 3’ end, and is known

as the poly-A tail.

These ends help when the mRNA is being attached to the ribosome, and also help in protecting the sequences

from any hydrolytic enzymes in the cytoplasm.

The untranslated regions (UTRs) are sequences of code that are not later translated, but still serve some

purpose. There is a 5’ UTR near the 5’ end and a 3’ UTR at the 3’ end. In between are the codons that are to be

translated.

Split Genes and RNA splicing:

RNA splicing is the process by which certain unnecessary code is removed.

There are often segments such as UTAs that do not code for anything that are spread across the sequence.

These are called introns. Exons, is the code/are the codons that are to be translated or expressed.

Spliceosomes are a complex of proteins and other molecules that carry out the splicing. It involves removing

introns and putting back together exons so that they are continuous.

Thus, in RNA splicing introns are removed, and the resulting final mRNA molecule leaves the nucleus and is ready

to be translated.

Some genes are able to express more than one polypeptide. They do this by switching which sequences are the

exons and which sequences are introns, thus changing what will be translated into amino acids and proteins. This is

known as Alternative RNA splicing.

 Ribozymes:

Ribozymes are RNA molecules that can act as enzymes. Some organisms in nature that are eukaryotic have their

pre-mRNA act as spliceosomes. What this means is that the mRNA strand splices itself.

RNA is single stranded so it can form a 3D shape like a protein by binding with itself in places.

Some of the bases may also have functional groups that allow for extra function. They are also able to bind to

specific substrates (A property of enzymes).

14.4 Translation is the RNA-directed synthesis of a polypeptide - A closer look:

Molecular components of Translation:

tRNA is what acts as the translator between codons and amino acids. There lie amino acids around the

cytoplasm and while reading the codons, the tRNA synthesizes a polypeptide accordingly.

The Structure and Function of Transfer RNA:

The tRNA consists of nucleotides that are bended and bond with each other to form a distinct L shape. tRNA

have one end that can read codons and another end that can attach polypeptides.

The anticodon is the complementary sequence to the codon, and is how the tRNA ‘reads’ the bases. The amino

acid attaches to the 3’ end of the molecule.

tRNA molecules bind to amino acids through the family of enzymes known as aminoacyl-tRNA synthetases. There

are 20 of them for each amino acid that is to be attached to the tRNA molecule. Then, the tRNA is able to go to the

ribosome and attach the amino acid based on the codon.

There are not as many tRNA molecules as there are codon possibilities. There is some versatility with what

codons the different tRNA can bind with. This is known as wobble.
Ribosomes:

Ribosomes are structures that allow tRNA anticodons and mRNA codons to couple with each other. It consists of

two subunits: the large and the small subunits. Each of these subunits are made up of rRNA (ribosomal RNA) and

proteins.

In Eukaryotes the ribosomes are synthesized in the nucleus and are assembled then leave the nucleus.

Bacterial and Eukaryotic ribosomes are similar, but do have differences. Bacterial ribosome inactivation is the

key to many antibiotics.

There is evidence that supports that rRNA is what causes the ribosome to hold its shape rather than the

proteins.

Within the ribosome are three sites:

The tRNA with the appropriate amino acid enters the ribosome through the A site. The tRNA in the P site holds

the polypeptide chain and following amino acids bond to the carboxyl groups of other amino acids by forming peptide

bonds.

Finally, the tRNA leaves through the E site or exit site.

Building a Polypeptide:

Translation is also broken down into these stages like transcription.

Ribosome Association and Initiation of Translation:

In Eukaryotes, first the mRNA and small ribosomal subunit come together. (At this point the ribosome has

not been assembled). Then, the tRNA attached to the amino acid Met (This is specifically known as the initiator

tRNA) starts at the 5’ cap and scans until it finds the start sequence AUG.

Once this is done, the large ribosomal subunit is attached, and a new tRNA is allowed to enter through the

A site and replace the last tRNA while adding to the polypeptide chain.
This process that brings together all of these components into the translation initiation complex is powered by the

hydrolysis of GTP and initiation factors.

The Met protein is known as the N terminus since the amino group is exposed. The chain adds until the final

protein, and that protein is the C terminus since its carboxyl group is exposed. Protein synthesis always occurs in

this N → C direction.

Elongation of the Polypeptide Chain:

Elongation of the polypeptide chain happens at the C terminus of the preceding amino acid in three steps.

The codon is first recognized and one molecule of GTP is hydrolyzed here for accuracy.

In peptide bond formation, a peptide bond is formed by first removing the polypeptide chain from the previous tRNA,

moving the new tRNA to the P site, attaching the amino acid to the C terminus, and attaching the polypeptide back

onto the tRNA.

(Technically this step and the previous step occur simultaneously.) Another GTP is hydrolyzed so that one

tRNA moves to the E site and the new tRNA moves to the P site. Also, a new tRNA is brought to the A site.

Termination of translation:

At the end of the mRNA, there are the stop sequences that do not actually translate to amino acids, but

instead signal a release factor to bind to the stop codon.

Then, a water molecule is attached to the polypeptide and it is released.

Afterwards, other proteins help to disassemble the translation initiation complex.

Completing and Targeting the Functional Protein/Protein Folding and Post-translational Modifications:

The gene determines the amino acid sequence which also determines the 3D shape of a protein that is

synthesized. There are helper proteins that fold proteins in the right ways to aid in the correct folding of the

molecule.
 In post-translational modification, the amino acids may be modified or removed, the polypeptide may be

cleaved, etc. There are certain processes like these that the protein require in order to activate. This is also a

method of protein regulation so that the proteins are not active immediately.

Targeting Polypeptides to Specific Locations:

There are free and bound ribosomes; those that are in the cytosol and those that are attached to rough

ER and are part of the endomembrane system. They have the same structure but are positioned to produce

different kinds of proteins.

All protein synthesis begins in the cytosol, however. Signal peptides are amino acids on the polypeptide that

is being created that signal for the ribosome to attach to the ER and become bound. The proteins that recognize

the signal peptides are proteins of the translational complex known as signal-recognition particles or SRP.

The polypeptide continues elongating into the lumen, and is transported to wherever it needs to go through

vesicles.

Other organelles in all organisms use such protein signaling techniques and are highly specialized to suit the

function and destination of a protein.

Making Multiple Polypeptides in Bacteria and Eukaryotes:

There is always a need for more than just one molecule of a polypeptide in both bacteria and eukaryotes.

The mRNA is simply reused continuously by passing through multiple ribosomes so that polypeptide chains can grow

simultaneously. These are known as polyribosomes or polysomes.

In bacteria, more mRNA can just be produced. This is a bit more tedious in Eukaryotic cells since they go

through a refinement step.

 14.5 Mutations of one or a few nucleotides can affect protein structure and function:
- Mutations are changes in genetic information and sequences in DNA. There are large mutations, and smaller
scaled ones. Point mutations are mutations that affect only single nucleotide pairs. It is important to
note that all genetic diversity and phenomena such as speciation arise from genetic mutations, at least on
the large evolutionary scale.
- Mutations to genes in a parent that are permanent are transferred to the offspring. If the
mutation causes adverse effects in the parent, it may not be able to reproduce at all, but if it
does it passes on a genetic disease. These are usually homozygous dominant or recessive, but can
sometimes still be harmful since a heterozygous individual for the disease may produce an
offspring that is homozygous for the disease.
- Conditions such as sickle-cell anemia and familial cardiomyopathy are examples of hereditary
diseases that are caused by point mutations.
- Types of Small-Scale Mutations:
- Substitutions:
- In Nucleotide-pair substitution, a nucleotide and its pair are replaced by another pair.
These changes can be silent mutations (they can have no effect on the protein that is
encoded by the affected gene). Missense mutations are mutations caused by replacement
that change an amino acid of a protein. This often does not have much effect as well.
Nonsense mutations arise when a substitution leads to the code being interpreted as a
stop codon. Here, the translation would end prematurely, and the rest of the protein will
not be translated. A frameshift mutation is a genetic mutation caused by a deletion or
insertion in a DNA sequence that shifts the way the sequence is read.
Largest impact: Nonsense mutation or frameshift mutation

15.2 Eukaryotic gene expression is regulated at many stages:

In all organisms and cells, the entire genome of the organism is contained within the nucleus of the cell. The

specialization of the cell comes from the special proteins that it produces to shape itself/secrete etc. These proteins
come from speciifc parts of the genome, but a specialized cell will never need the entire genome to transcribe and

translate. Gene regulation is used to turn genes on or off in order to give specialization to a cell.

Differential Gene Expression:

The phenomenon mentioned above is known as differential gene expression. They all have the same genes, but different

genes are expressed. The inability to do this is the premise of several diseases that can be genetic.

Gene regulation is not only done by cutting off the source, however. The entire process of DNA → RNA → polypeptide

has several stages where it can be cut off. If at any time it is cut off, no polypeptide will be produced, and so there

the gene is regulated to some extent.

The most common gene regulation stage in all organisms is the transcription stage. This regulation is usually triggered

by hormone pathways and other external signals.

Regulation of Chromatin Structure:

There are several levels of DNA structure so that they can be compact and fit within the nucleus, but also so that

some genes can be condensed and entirely cut off so that they are not expressed and are in effect ‘cut off’.

Histone modification and DNA Methylation:

Chemical modifications done on histones help in regulation of DNA. The N terminuses of the histones protrude outward

so that they can receive and be modified by functional groups.

When an acetyl group is added, the histone is acetylated, and the gene is expressed. When a methyl group is added, the

histone is methylated, and the gene is suppressed.

Generally however, the DNA itself is methylated in DNA methylation where the DNA is directly suppressed instead of

the histones.

Methylation is done in cells so that they are specialized and so that they have specific function. Because of this, when

specialized cells divide, the daughter cells often inherit the methyl groups so that they are also specialized.
Epigenetic Inheritance:

Epigenetic inheritance is where regulation of DNA and genes is done by not directly affecting the DNA sequences.

This is the basis for several diseases that throw the methylation out of order since there are enzymes that regulate

this. If this is out of balance, the function of the cell and the proteins that it produces are also out of balance.

Regulation of Transcription Initiation:

What the above processes do is affect the chromatin structure. Affecting the chromatin structure physically does not

allow the RNA polymerase to go through with transcription. This is the first level of gene regulation.

The next stage in both prokaryotes and eukaryotes is control of the stages of DNA transcription and translation.

Since there are more stages in Eukaryotes, there are more stages where it can be potentially regulated.

Organization of a typical Eukaryotic cell:

This is review from chapter 14.

There is a transcription factor complex consisting of RNA polymerase II and other proteins. These go upstream on a

gene and produce a pre-mRNA. Then the caps are added and it is spliced

The way that these are regulated are through noncoding control elements which are strips of nucleotide sequences

that allow transcription factors to bind to them and block the RNA polymerase from going through with transcription.

The roles of transcription factors:

In eukaryotes, general transcription factors are transcription factors that are required by all genes for transcription

to begin. The TATA box is a fairly standard code on all genes, but besides this, general transcription factors are

proteins that assemble into the transcriptional protein complex with the help of protein protein interactions.

This only initiates the process, but there are other factors mentioned below that speed up the process and are

gene-specific.

Enhancers and Specific Transcription Factors:

Specific Transcription Factors are specific to the gene and can speed up or slow down the transcription rate.

Proximal control elements are placed near the promoter, and work closely with the polymerase.

Enhancers consist of distal control elements that are farther downstream on a gene. They can either be nucleotide

sequences or proteins, but in either case, they are usually specific to the gene that they are on.

Sometimes, the gene is actually bent with a protein so that the enhancer and promoter can meet. There is a mediator

protein that allows a formation of a complex such that the enhancer can affect the promoter although it is much

further downstream.

In Eukaryotes, there are several methods by which repressors can function. They can block specific code as seen

before in the operon, and stop RNA polymerase from moving on. They can also block activators from binding. Although

this goes back to the methylation concepts, some repressors are able to affect the histone structure as well in

eukaryotic cells.

Combinatorial Control of Gene Activation:

In the enhancers mentioned above, there are sequences of distal control elements. In organisms, although there are

several different needs for regulation through those control elements in different genes, there are only a few types

of control elements. It is more the combination of control elements and transcription factors in the enhancer that

give it speciality.

Furthermore, the transcription factors that attach to the control elements need to do so in a specific way in order to

inhibit or activate the gene. This, in effect, is like a unique key and lock.

Coordinately Controlled genes in eukaryotes:

When there are several genes that have proteins that function together in the cell, in prokaryotes, they are placed

in operons.
In eukaryotes however, genes are spread over different chromosomes. There are control factors that allow for

simultaneous transcription of the genes involved.

Hormones and steroid are what cause this coordination and act as signals. When one of these molecules enter the

nucleus, a hormone-receptor complex forms and acts as an activator for transcription for those genes.

There are also signal-induction pathways that trigger the activation of transcription from outside of the cell.

Mechanisms of Post-Transcriptional Regulation:

Again, there are more stages after transcription which means that there are more stages for regulation.

RNA processing:

Alternative RNA splicing is the process by which multiple final mRNA molecules can be produced from one pre-mRNA

strand by switching introns and exons and thus changing the meaning of the RNA each time.

Alternative DNA splicing is used for about 90% of the human genome. This reduces the need for multiple genes and

keeps the genome relatively short but concise.

mRNA degradation:

The lifespan of an mRNA in the cytoplasm controls whether or not there is an RNA to produce proteins. In bacteria,

the mRNA last a short time, but this lifetime can change dynamically according to the conditions. In eukaryotic cells, it

depends on the caps attached to the poly-A tail.

Initiation of Translation:

Translation can be regulated if either of the caps of the mRNA are blocked by some factor so that it cannot pass

through the ribosome.

Other methods in eggs involve not attaching tails to mRNA and leaving them in the nucleus until necessary.
Sometimes, the mRNA are kept in the nucleus until initiation factors enable a cell-wide release and production of

protein.

Protein processing and degradation:

After proteins are produced, cleaving the protein/polypeptide or chemically altering the protein to give it a 3D shape

can activate/deactivate the proteins. This is a step of regulation because cleaving, for example, can be held off until

it is necessary.

Certain proteins are only activated when they reach their destination. This is another point for regulation.

The lifetime of proteins is heavily regulated as a main process of regulation of the cell. Ubiquitin is often attached to

proteins to mark them for destruction.