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What proteins are associated with DNA?

How are proteins involved in transcription?

How is protein production controlled?

Why is it important that protein production is controlled?

Why is protein structure important in relation to its function?

DNA AND PROTEINS

This lesson will cover• DNA and its

associated proteins• Other proteins

involved with transcription

DNA AND PROTEIN ASSOCIATION

DNA AND PROTEIN ASSOCIATION

• DNA binds to a number of proteins.• Positively charged histone proteins bind to the

negatively charged sugar-phosphate backbone of

DNA in eukaryotes.• DNA is wrapped around histones to form nucleosomes

packing the DNA in chromosomes.

DNA AND PROTEIN ASSOCIATION

Animation

HISTONE PROTEINS AND NUCLEOSOME

OTHER DNA PROTEINS AND LIGAND BINDING

• Other proteins have binding sites that are specific to

particular sequences of double stranded DNA.• When this happens they can stimulate or inhibit the

initiation of transcription.

Animation

DNA AND PROTEIN COMPLEX IN TRANSCRIPTION

TRANSCRIPTION FACTORS

• Transcription factors (TFs) are molecules involved in regulating gene expression.

• They are usually proteins, (they can be short, non-coding RNA). • TFs are also usually found working in groups or complexes,

forming multiple interactions that allow for varying degrees of control over rates of transcription.

TRANSCRIPTION FACTORS

• In people (and other eukaryotes), genes are usually in a default "off" state, so TFs serve mainly to turn gene expression "on".

• TFs work by recognizing certain nucleotide sequences (motifs) before or after the gene on the chromosome.

• The TFs bind, attract other TFs and create a complex that eventually facilitates binding by RNA polymerase, thus beginning the process of transcription.

BINDING CHANGES THE CONFORMATION OF A PROTEIN

• Proteins including enzymes are three-dimensional and have a specific shape or conformation.

• As a ligand binds to a protein binding site, or a substrate binds to an enzyme’s active site, the conformation of the protein changes.

• This change in conformation causes a functional change in the protein and may activate or deactivate it.

BINDING TO LIGANDS

• A ligand is a substance that can bind to a protein. • R groups not involved in protein folding can allow

binding to these other molecules. • Binding sites will have complementary shape and

chemistry to the ligand.• The ligand can either be a substrate or a molecule that

affects the activity of the protein.

All chemical reactions require energy to enable them, this is the activation energy.

Enzymes lower the activation energy.

2 types of reaction are:

Anabolic (synthesis) a dehydration synthesis reaction.

Catabolic (degradation) a hydrolysis reaction.

Enzymes

Anabolic Reactions

• Uses energy to SYNTHESISE large molecules from smaller ones e.g. Amino Acids Proteins

• Also known as endothermic reactions

ENDOTHERMIC REACTION

Catabolic Reactions

• These release energy through the BREAKDOWN of large molecules into smaller units e.g. Cellular Respiration: ATP ADP + Pi

• Also known as exothermic reactions

EXOTHERMIC REACTION

Enzyme types

Proteases - break down proteins into amino acids by breaking peptide bonds (hydrolysis).Nucleases - break down nucleic acids into nucleotides (hydrolysis).

ATPases - hydrolysis of ATP.

Kinases - add phosphate groups to molecule.Phosphatases – remove phosphate groups

Control of Enzyme activity

Control of enzyme activity occurs in these ways• number of enzyme molecules present

• compartmentalisation• change of enzyme shape by

competitive inhibitors, non-competitive inhibitors,

enzyme modulators, covalent modification

• end product inhibition

How do enzymes work?

Induced fit and enzymes• Enzymes are not necessarily a perfect sit to substrate• The enzyme changes shape in response to close

association with the substrate.• This the Induced fit theory

A molecule close to shape of substrate competes directly for active site so reducing the concentration of available enzyme.

This can be reversed by increasing the concentration of the correct substrate unless the binding of competitor is irreversible.

Competitive inhibition

Succinate dehydrogenase catalyses the oxidation of succinate to fumarate (respiration)Malonate is the competitive inhibitor

Malonate example

An inhibitor binds to the enzyme molecule at a different area and changes the shape of the enzyme including the active site.

This may be a permanent alteration or may not.

Non-competitive inhibition

•Inhibition can either be reversible or non-reversible•Some inhibitors bind irreversibly with the enzyme molecules.

•The enzymatic reactions will stop sooner or later and are not affected by an increase in substrate concentration.

•Irreversible inhibitors include heavy metal ions such as silver, mercury and lead ions.

Some enzymes change their shape in response to a regulating molecule.

These are called allosteric enzymes

Positive modulators (activators)

stabilise enzyme in the active form.

Negative modulators (inhibitors)

stabilise enzyme in the inactive form.

Enzyme modulators

Allosteric Enzymes

Involves the addition, modification or removal of a variety of chemical groups to or from an enzyme (often phosphate.)

These result in a change in the shape of the enzyme and so its activity.

These include phosphorylation by kinases and dephosphorylation by phosphatases.

Conversion of inactive forms to active forms e.g. trypsinogen and trypsin

Covalent modifications

An example of activation is trypsinogen to trypsin

trypsinogen activated by enterokinase in duodenum

Trypsin is synthesised in the pancreas, but not in its active form as it would digest the pancreatic tissue•Therefore it is synthesised as a slightly longer protein called TRYPSINOGEN•Activation occurs when trypsinogen is cleaved by a protease in the duodenum•Once active, trypsin can activate more trypsinogen molecules

Often seen in pathways that involve a series of enzyme controlled reactions.

The end product once produced has an inhibiting affect on an enzyme in the reaction.

Example:

Bacterial production of amino acid isoleucine from threonine.

5 stages enzyme controlled

Threonine Isoleucine

End product Inhibition

To summarise

As a ligand binds to a protein or a substrate binds to an enzyme’s active site, the conformation of the protein changes,This change in conformation causes a functional change in the protein.

To summarise

In enzymes, specificity between the active site and substrate is related to induced fit.When the correct substrate starts to bind, a temporary change in shape of the active site occurs increasing the binding and interaction with the substrate.The chemical environment produced lowers the activation energy required for the reaction.Once catalysis takes place, the original enzyme conformation is resumed and products are released from the active site.

To summarise

In allosteric enzymes, modulators bind at secondary binding sites.The conformation of the enzyme changes and this alters theaffinity of the active site for the substrate.Positive modulators increase the enzyme affinity whereasnegative modulators reduce the enzymes affinity for the substrate.

Haemoglobin and Oxygen

COOPERATIVITY IN HEMOGLOBIN

Deoxyhaemoglobin has a relatively low affinity for oxygen.

As one molecule of oxygen binds to one of the four haem groups in a hemoglobin molecule it increases the affinity ofthe remaining three haem groups to bind oxygen.

Conversely, oxyhaemoglobin increases its ability to looseoxygen as oxygen is released by each successive haem group.

This creates the classic sigmoid shape of the oxygendissociation curve.

DISSOCIATION CURVE OF HAEMOGLOBIN

Deoxyhaemoglobin Oxyhaemoglobin

Disassociation releasing oxygen to tissues

Association binding oxygen in lungs

EFFECTS OF TEMPERATURE AND PH

• Low pH = low affinity.

• High temperature = low affinity.

Exercise increases body temperature and produces more

CO2, acidifying the blood.

This has a corresponding effect on the oxyhaemoglobin

dissociation curve.

Sickle Cell AnaemiaLow oxygen levels cause change in haemoglobinstructure.Strands cause cells to takeon bent sickle shapeblocking capillaries.

HIGH ALTITUDE AND OXYGENThe concentration of oxygen (O2) in sea-level air is 20.9%, so the

partial pressure of O2 (pO2) is 21.136 kPa.

Atmospheric pressure decreases exponentially with

altitude while the O2 fraction remains constant to about

100 km, so pO2 decreases exponentially with altitude as well.

It is about half of its sea-level value at 5,000 m (16,000 ft), the

altitude of the Everest Base Camp, and only a third at 8,848 m

(29,029 ft), the summit of Mount Everest. When pO2 drops, the

body responds with altitude acclimatization.

BBC Horizon How to kill a Human Being

To summarise

Some proteins with quaternary structure show cooperativityin which changes in binding alter the affinity of the remainingsubunits.Cooperativity exists in the binding and release of oxygen inHaemoglobin.Temperature and pH influence oxygen association.