DNA and Proteins

DNA

• Stores and transmits genetic information.

• Functions in the same way in all living things.

• Helical double-stranded molecule.

• Strands are antiparallel, if one strand is 5’ to 3’ then the other is 3’ to 5’.

• Humans read in the 5’ to 3’ direction.

Nucleotide

• Subunit that makes up DNA.

• Consists of a phosphate and sugar backbone attached to a base.

DNA Bases

Guanine, Cytosine, Thymine, and Adenine.

Eukaryotic Cells

• Multiple linear chromosomes.

• DNA is bound to histones.

• DNA is stored in nucleus.

• No extra chromosomal DNA.

• Unbound circular DNA in mitochondria and chloroplasts.

• Chromosomes attach to spindle fibres during cell division.

Prokaryotic Cells

• Single circular chromosome.

• DNA is unbound.

• DNA is found in the cytoplasm.

• May contain additional chromosomal DNA (plasmids).

• Does not contain organelles with own DNA.

• Chromosome attaches to cell membrane during cell division.

Semi-Conservative DNA Replication

• DNA strands are separated and complementary base pairs are added to each of the original (template) strands.

• DNA polymerase can only synthesise in the 5’ to 3’ direction.

Gene

Segment of DNA consisting of a unique sequence of bases that code for a protein or RNA molecule.

Chromosome

Unit of DNA that contains many genes.

Enzymes

• Biological catalysts that speed up metabolic reactions without being consumed.

• Are proteins.

• Have an active site that has a specific 3D shape complementary to that of a substrate.

Induced-Fit Model

1. Complementary substrate binds to active site forming an enzyme-substrate complex.

2. Active site changes shape slightly, inducing a tighter fit, placing stress on bonds and lowering activation energy.

3. Resulting product or products are released, the enzyme returns to normal shape and can be used again.

Activation Energy (E_A)

The initial input of energy required for a reaction to begin.

Enzymes in Breakdown Reactions

Lower the activation energy by the induced fit between substrate and enzyme, which places stress on chemical bonds, making them easier to break.

Enzymes in Synthesis Reactions

Lower the activation energy by the induced fit between substrate and enzyme, increasing the chance of 2 or more substrate molecules colliding and facilitating the correct orientation for successful collisions.

Factors that Affect Enzyme Activity

Decreasing activity:

• Low temperature, molecules move slower and so less successful collisions between enzyme and substrate molecules.

• High temperature, enzymes are denatured.

• Low pH or high pH, enzymes are denatured.

Increasing activity:

• Increasing substrate concentration, increases number of successful collisions. Reaches point of saturation when all enzyme active sites are occupied.

• Increasing enzyme concentration, increases number of successful collisions. This is assuming there is an infinite supply of substrate.

Inhibitors

Chemicals that disrupt the enzyme’s active site.

Competitive Inhibitors

• Have a complementary shape to the enzyme.

• Bind to the active site, preventing substrate from binding.

Non-Competitive Inhibitors

• Bind to the enzyme (not in active site), altering the active site shape.

• Substrate no longer complementary, preventing it from binding.

Proteins

Have a specific 3D shape determined by their amino acid sequence that allows them to carry out a specific function.

Protein Structure

1. Primary Structure

2. Secondary Structure

3. Tertiary Structure

4. Quaternary Structure

Primary Structure

The sequence of amino acids in a polypeptide chain, determined by the series of bases on mRNA, which is in turn determined by the unique DNA base sequence of the gene.

Secondary Structure

The coiling or folding of sections of the polypeptide chain into helices and sheets, determined by the primary structure and held together by hydrogen bonds.

Tertiary Structure

The 3D shape of a single polypeptide chain, determined by the secondary structure.

Quaternary Structure

The 3D shape of two or more polypeptides, determined by interactions between multiple polypeptides, enabling the formation of chemical bonds.

Protein Synthesis

The flow of genetic information in a cell. DNA to mRNA via transcription, and mRNA to proteins via translation.

RNA vs. DNA

RNA:

• generally single stranded.

• contains base uracil.

• contains sugar ribose.

• temporary store of genetic information.

DNA:

• double stranded.

• contains base thymine.

• contains sugar deoxyribose.

• permanent store of genetic information.

Transcription

The process in which genetic information in a gene is transcribed to mRNA.

• The part of the DNA to be copied unwinds, the two strands separate, and RNA nucleotides assemble at the template DNA strand to form an mRNA molecule.

• Main enzyme involved is RNA polymerase.

• mRNA carries genetic message from DNA in the nucleus to ribosomes in the cytoplasm.

Translation

Genetic information on the mRNA is translated into proteins.

• mRNA attaches to the ribosome.

• A tRNA molecule carrying a specific amino acid attaches to the 3-base codon on the mRNA via complementary base pairing with the 3-base anticodon on tRNA.

• the ribosome (containing rRNA) joins amino acids together by peptide bonds to form a polypeptide.

• The polypeptide folds into a unique 3D shape to form a functional protein.

rRNA

Ribosomal RNA.

mRNA

Messenger RNA.

tRNA

Transport RNA.

Protein Synthesis Location

In eukaryotic cells:

• transcription in nucleus

• translation in ribosomes in the cytosol

In prokaryotic cells:

• transcription and translation in the cytosol

Splicing

In eukaryotes, sections of RNA are removed before leaving the nucleus. Both exons and introns are transcribed to pre-mRNA.

Introns

Non-coding sequences, are removed in splicing.

Exons

Coding sequences, are joined together in splicing then exit the nucleus to be translated and expressed.

Ribosomes

• Found in all cells, located in the cytosol either free-floating or attached to the rough endoplasmic reticulum.

• Sites of protein synthesis.

• Composed of protein and RNA, the RNA is called ribosomal RNA and is produced in the nucleolus of cells.

• The function of rRNA is to catalyse the formation of peptide bonds between amino acids during translation.

Differentiation

• All cells start as stem cells with identical DNA.

• Differentiation is the process in which cells become specialised in structure and function.

• The DNA remains identical.

Stem Cells

Undifferentiated cells that can produce, by mitotic division, daughter cells that can differentiate.

Gene Expression

Cellular differentiation is controlled by gene expression, which is the process where a gene is transcribed to form RNA.

• Specific genes of certain cells are expressed to produce specific gene products (proteins).

• Other genes are not expressed, thus not producing the gene products.

• The specific set of proteins give rise to a cell with specialised structure and function.

Phenotypes

Observable (and biochemical) characteristics resulting from genotype.

Genotype

The particular set of genes (alleles) and organism has for a trait.

Factors that Affect Gene Expression

Environmental factors, products of other genes (proteins and RNA), DNA methylation, and histone modification.

Gene Expression - Environmental Factors

Such as temperature and chemicals.

Gene Expression - Products of Other Genes (Proteins and RNA)

• Transcription factors are regulatory proteins that control gene expression. This includes proteins and RNA.

• Activator proteins switch genes ‘on’ by binding to DNA promoter region, activating transcription.

• Repressor proteins switch genes ‘off’ by blocking the attachment of RNA polymerase, preventing transcription.

• Some gene products can prevent translation by binding to mRNA making it unable to successfully bind to the ribosome.

• mRNA can be degraded by the gene product small-interfering RNA, preventing it from being translated.

Gene Expression - DNA Methylation

• Methylation of DNA cytosine nucleotides.

• The addition of a methyl group to cytosine nucleotides can deactivate genes (other forms of epigenetics can activate genes).

• Increased DNA methylation of a gene can prevent RNA polymerase from binding and thus transcribing the gene, reducing gene expression.

Gene Expression - Histone Modification

• By modifying histones, cells can regulate which genes are turned on or off, meaning whether certain genes are expressed or not.

• DNA loosely packaged around histones is easily accessible therefore easier to be expressed.

• DNA tightly packaged around histones is harder to be expressed.

• Amino acid chains extend from histone protein, called tails. Histones can be modified by attaching ‘chemical tags’ to their tails.

Epigeneitcs

• Long-term inheritable changes in gene expression that don’t involve changes in the DNA sequence.

• Can be passed on by daughter cells.

• Distinguishes identical twins.

Epigenetic Changes Leading to Cancer

• Deactivating tumour suppressor genes (genes that prevent cell division).

• Activating oncogenes (genes that promote cell division).

Mutations

Random and permanent changes in the sequence of nucleotides in DNA. Spontaneous mutations usually occur during:

• DNA replication

• cell division

Can be induced by:

• ionising radiation

• mutagenic chemicals

• some viruses

Mutations During DNA Replication

1. DNA polymerase mismatches DNA complementary base pairs.

2. Leading to insertion of incorrect base (sometimes is repaired).

3. Upon further replication, mismatched base is permanently changed in the DNA base sequence on both strands.

Chromosomal Mutations

• Errors occur during cell division (mitosis or meiosis).

• Chromosomes do not separate correctly.

• Cells can receive extra or missing chromosomes.

Mutagens

Physical or chemical factors that increase the rate of mutation in DNA. Examples include:

• ionising radiation (e.g. UV radiation, x-rays).

• mutagenic chemicals (e.g. chemicals, cigarette smoke).

• viruses (mutations can occur if some of the viral DNA becomes incorporated into the host cell DNA).

Substitution Mutation

A base in the sequence is replaced by a different base.

Deletion Mutation

A base is removed from the sequence, causing a frameshift.

Insertion Mutation

A base is inserted into the sequence, causing a frameshift.

Frameshift

All bases in an mRNA strand are shifted from the point of insertion/deletion. Alters many amino acid sequences, leading to altered folding of protein and possibly making it non-functional.

Silent Mutation

A single DNA base mutation does not change the amino acid produced.

Somatic Cells

Cells that do not lead to gametes. They are body cells.

Germ-Line Cells

Cells that divide by meiosis to produce gametes. They are sex cells.

Gametes

Sex cells (sperm or egg).

Somatic Mutations

• Mutation occurs in somatic cell (body cell) and is therefore confined to the organism.

• The mutation will only be present in the cell and any cells arising by mitosis.

• This may result in cancer.

• Cannot be passed on to offspring.

Germ-Line Mutations

• Mutation occurs in a germ-line cell (sex cell) and therefore mutation will be present in every cell of the organism.

• Can be passed on to offspring.

• Results in absent or altered proteins in offspring and potential diseases.

Inheriting Genetic Abnormalities/Diseases

Offspring won’t always inherit inheritable disease from an affected parent, usually an element of chance involved.

DNA Extraction

• Requires cell membrane to be broken, releasing contents of the cell.

• Enzymes can then be used to remove proteins, including histones from the DNA.

• DNA can then be isolated, amplified and then analysed.

Polymerase Chain Reaction (PCR)

Takes small amount of DNA and copies many times.

1. Heat DNA to break weak hydrogen bonds between strands, exposing bases.

2. Add primers and cool. Primers bind to single strands, preventing them from rejoining and providing a starting point for DNA synthesis by DNA polymerase.

3. Heat. Add free nucleotides and heat-tolerant DNA polymerase which joins the free nucleotides with the original strands.

Primers

Short single strands of DNA and RNA.

Electrophoresis

Used to create DNA fingerprint.

1. Collect cells.

2. Extract DNA.

3. Cut DNA into fragments using specific restriction enzyme.

4. Seperate fragments using gel electrophoresis. DNA samples are loaded into wells at the negatively charged electrode. When electrical current is applied, DNA will move toward positively charged electrode due to the slight negative charge of DNA. Smaller fragments will move further than larger fragments.

Restriction Enzymes

Used to cut DNA samples into different length fragments, and to cut DNA at a specific complementary location.

DNA Sequencing

The process of determining the DNA base sequence for a particular piece of DNA or an entire genome of a particular organism or species.

Electropherogram

A graphical representation of the results of electrophoresis. y-axis (peak hight) represents amount of PCR product, x-axis represents fragment size.

Short Tandem Repeats (STRs)

• Long stretches of DNA made up of repeated nucleotide sequences within the non-coding regions of an individual’s genome.

• Can be cut using restriction enzymes then separated with gel electrophoresis for comparison.

• Longer repeats will generate larger fragments, shorter repeats will generate smaller fragments.

DNA Profiling

1. DNA sample is obtained, extracted and multiplied using PCR.

2. DNA sample is cut using specific restriction enzyme, producing DNA fragments of different size unique to the individual.

3. Fragments are analysed using gel electrophoresis, which separates the fragments of known size giving a unique banding pattern.

4. Electrophoresis bands of known size are used to create DNA profile, displayed as an electropherogram.

Ethical/Cultural Considerations of DNA Profiling

• Compulsory acquisition of genetic information could be seen as an abuse of human rights.

• Individuals could face discrimination if genetic information was acquired and misused by insurance companies or employers.

• May conflict with religious ideologies.

• Some individuals may not consent to invade practices to collect genetic information on cultural grounds.

Economic Considerations of DNA Profiling

• Large set up costs to collect, store and maintain genetic information, likely increase taxes to fund, putting pressure on government budgets.

• Crime prevention will have economic benefits.

• Targeted health for individuals has economic benefits, increases productivity, and reduces burden on health system.

Genetic Engineering

The process of combing DNA from two different origins to produce recombinant DNA, creating a genetically modified organism (GMO), also known as a transgenic organism.

Genetic Engineering Process

1. Extract DNA from cells.

• cells broken apart to release contents (using mild heat and detergent)

• separation of DNA from contents of cell

2. Select DNA segment of interest, using labelled probes.

• made of singe stranded DNA or RNA

• complementary to a section of target gene

• labelled with radioactive or fluorescent markers

3. Remove DNA segment of interest.

• using specific restriction enzymes that cut specific nucleotide sequences of DNA

• can produce blunt or sticky ends that recombine only with complementary ends (result from different restriction enzymes)

4. Transfer DNA into organism.

• microinjection

• viral vectors

• bacterial plasmids (used for plants)

• yeast

• Electroporation

Transferring DNA via Microinjection

1. The specific genes are selected and removed using probes and restriction enzymes.

2. The DNA containing the gene of interest is injected directly into the cell nucleus (usually zygote) using a very fine micropipette.

Transferring DNA via Viral Vectors

1. Gene of interest is removed using specific restriction enzyme.

2. Viral DNA is cut using same specific restriction enzyme.

3. Gene of interest inserted into the viral DNA, will join due to complementary base pairing.

4. Virus is used as a vector to infect cells of the host organism. When virus integrates its DNA into host, it also incorporates the gene of interest into the host DNA.

Transferring DNA via Bacterial Plasmids (Plants)

1. Gene of interest is removed from a cell using specific restriction enzyme.

2. Bacterial plasmid is cut using the same specific restriction enzyme to produce complementary sticky ends.

3. Gene and plasmid are mixed together and join due to complementary base pairing. Enzyme DNA ligase seals the breaks in DNA.

4. Plasmid is instead back into bacteria.

5. Bacteria can be used to ‘infect’ plant, which will directly incorporate its own DNA, including gene of interest, into DNA of plant cells.

Plasmid

Circular DNA molecule found in bacteria, seperate from the chromosomal DNA.

Transferring DNA via Yeast

• Yeasts are unicellular eukaryotic organisms that have plasmids that can be modified like bacterial plasmids.

• An advantage is that since it is a eukaryote, the entire eukaryotic gene (both introns and exons) can be inserted.

Transferring DNA via Electroporation

• An electrical pulse is used to deliver DNA directly into cells.

• Pulse induces temporary pores in cell membrane.

• Cell membrane reseals and is left unharmed.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)

• An adaptive immune system of bacteria.

• When a virus attacks bacteria, it stores a copy of the virus DNA. This can be transcribed to RNA, loaded into an enzyme called Cas9, and used to cut the virus.

• The CRISPR/Cas9 system can be used to edit genes in live cells, through this process:

1. Guide RNA manufactured to be complementary to target DNA base sequence to be edited.

2. Guide RNA loaded into Cas9 protein.

3. Cas9 locates specific sequence complementary to guide RNA and cuts.

CRISPR - Disrupt

Single cut in DNA.

CRISPR - Delete

Two cuts in DNA using two different guide RNA.

CRISPR - Correct or Insert

Correct or insert a new gene by adding a DNA template alongside the CRISPR/Cas9 machinery.

New Protein Design

1. Design shape of protein.

2. Determine amino acid sequence.

3. Construct a gene to code for the desired amino acid sequence.

4. Gene is transferred to bacteria or yeast to produce new protein.

5. Cells are lysed and protein is isolated.

Uses of Protein Design

• Therapeutics

• Biosensors

• Agriculture

• Industrial enzymes

• Materials science