AOS 1 - Biology - Nucleic Acids and Proteins

Vocabulary flashcards from the lecture notes.

What are the steps of CRISPR-CAS9?

Exposure: Bacteriophage injects viral DNA, Cas 1 & 2 enzymes cut protospacer upstream from PAM. Protospacer injects into CRISPR gene as spacer.

Expression: CRISPR transcribed into guide RNA (gRNA) with repeats. gRNA attaches to Cas 9, forming CRISPR-Cas 9 complex.

Extermination: Complex scans for matching bacteriophage DNA; Cas 9 cleaves sugar-phosphate backbone to inactivate virus.

How is CRISPR-CAS9 used in genetic engineering?

Scientists create gRNA complementary to target DNA, bind to Cas 9, and insert into cell. Cas 9 finds PAM, DNA matches gRNA, and Cas 9 cuts selected DNA. Cell repairs blunt cut, effectively silencing the removed DNA's function.

What are Restriction Endonucleases?

Endonucleases are enzymes that cut DNA strands. Restriction enzymes are endonucleases that cut specific DNA locations by cleaving phosphodiester bonds of the sugar-phosphate backbone, creating sticky or blunt ends.

What are the properties of genetic code?

Rules defining how information in nucleotides (DNA/RNA) translates into functional molecules. DNA/RNA information is stored in three-sequence sections. A three-sequence on DNA is a triplet, transcribed to a codon on mRNA. tRNA has a complementary anticodon to the mRNA codon, with each codon coding for one amino acid.

What is the Polymerase Chain Reaction (PCR)?

A DNA manipulation technique that amplifies DNA by making identical copies, increasing the amount of DNA available.

What is Gel Electrophoresis?

Technique that organizes DNA fragments according to size. Restriction endonucleases cut DNA, samples are inserted into wells in agarose gel in buffer solution. Electric current sent through gel, DNA moves to positive end, smaller fragments move faster. Fragments stained with fluorescent dye and visualized under UV lamp.

Levels of Protein Structure

Primary: A single sequence of amino acids in a polypeptide chain.

Secondary: Alpha helix and beta pleated sheets, formed by interactions between backbones with the folding and coiling being formed by hydrogen bonds between different amino acids.

Tertiary: 3D structure of a polypeptide chain, with a combination of alpha helix and beta pleated sheets.

Quaternary: Comprised of multiple polypeptide chains, unlike other structures with one polypeptide chain.

What are the different Protein Functions?

• Storage

• Structural

• Hormone

• Enzyme

• Defense

• Transport

• Motor contractile functions.

  Determined by the structure, with tertiary structure allowing proper function. Monomers are amino acids (R-group, carboxyl group, carbon atom, amino group). Formed through condensation reaction.

Transcription

Helicase unwinds DNA, RNA polymerase attaches to promoter. RNA reads strand, creating the pre-mRNA. Transcription terminates at termination sequence. Pre-mRNA undergoes post-transcriptional changes to become mRNA.

RNA Processing

Involves 5-methyl cap to 5' end and poly-A tail to 3' end. Splicing removes introns and joins exons.

Translation

mRNA binds to ribosome, recognizes start codon. tRNA arrives with amino acid and complementary anticodons. tRNA reads strand, attaching to codons, amino acid joins polypeptide chain via peptide bonds until stop codon is reached, and the polypeptide chain is released.

Protein Secretory Pathway

Polypeptide released in ribosome. Rough ER folds and prepares protein. Vesicle buds off ER, travels to Golgi, which modifies/packages protein. Secretory vesicle buds off Golgi, leaves cell via exocytosis.

Ethical Approaches

Consequences-based: Aims to maximize benefit outcomes while reducing negative outcomes.

Virtues-based: Involves acting in accordance with virtues/morals of a good person, subjective approach.

Duty/Rules based: Aims to make decisions based on what is 'correct', aligns with rules.

Ethical Concepts

Integrity: Commitment to knowledge; acting truthfully and honestly.

Justice: Commitment to fairness; addressing each person equally and prioritizing the fair distribution of resources.

Beneficence: Commitment to maximizing benefits; acting in a way that promotes personal wellbeing and good for other people.

Non-maleficence: Commitment to minimizing harm; acting in a way that does the least harm to the most people.

Respect: Commitment to consideration; considering the values of others, including their welfare, freedom, autonomy, encourages providing people with the freedom to make their own decisions.

What occurs when denaturation occurs?

Creates a conformational change in the active site and decreasing the rate of reaction

What is a polypeptide chain?

A biomacromolecule made of a chain of amino acids folded into a 3D shape

What is a proteome?

The entire set of cells being expressed at a given time in an organism

Amino Acid Structure

Consists of: A R-group, amino group, carboxyl group and central carbon atom. Every part is the same in all amino acids – except for the R-group, this differentiates them and their function

DNA vs RNA

DNA: Deoxyribose sugar, double helix, thymine nucleotide base. - tRNA (transfer), mRNA (messenger), rRNA (ribosomal)

RNA: Ribose sugar, single helix, uracil nucleotide.

What are nucleotides?

The basic unit of a nucleic acid and consist of three components: Nucleotide bases (5 types possible), phosphate group, 5-carbon sugar (2 types possible).

What are nucleic acids?

Biochemical macromolecules that are made up of many nucleotide units. They include DNA and RNA.

Structure of Genes - Eukaryotes (DNA)

Consist of a promotor or TATA box, then introns and exons, and then the termination sequence

Structure of Genes - Prokaryotes (RNA)

Consist of a promotor region, then a operator region, which a repressor protein can bind to, to inhibit transcriptions of these genes. There are no introns, only exons, these are followed by a trailer and then the termination sequence.

Promotor Region

Found upstream, at the 5’ end, and is the binding site for the RNA polymerase – which allows the gene to be transcribed. Due to this, this region determines which genes are transcribed and the direction.

Operator Region

Is the binding site for the repressor protein in Prokaryote genes – which leads to the inhibition of gene expression.

Exons

DNA regions that are coding segments

Introns

DNA regions that are non-coding segments

Termination Sequence

A sequence of DNA that signals for the transcription of the gene to stop.

Alternative Splicing

During RNA processing the introns are spliced out because they consist of non-coding segments, this leaves the exons to be translated in gene expression. But sometimes the exons are removed, these are the coding segments, this leads to the pre-mRNA being able to produce many different mRNA molecules – depending on which exons are spliced out. Therefore, one gene can have the capacity to code for different mRNA strands that code for different proteins.

Gene Regulation

Gene regulation is the process of either inhibiting or activating gene expression. This ensures that organisms don’t waste energy on the unnecessary production of gene products such as proteins when they aren’t needed.

Regulatory Genes

The segments of DNA responsible for producing proteins that control the expression of other genes.

Structural Genes

A segment of DNA that codes for proteins that will be used functionally or structurally throughout the cell. These genes are not transcribed without the production of regulatory genes.

Operon

The operon consists of multiple structural genes that serve the same purpose and are often arranged into groups, in the same location, so that their function is controlled by a single promotor and operator. This becomes the operator region, where the repressor protein binds.

Binding to the operator region

If the operator regions is bound with a repressor -> RNA polymerase can’t move downstream from the promotor region -> inhibiting transcription of the gene. If the operator region is not bound with a repressor protein - > RNA polymerase can move downstream to the promotor region -> allowing the transcription of the gene

Trp Operon

Bacteria (prokaryotes), like E. coli, need amino acids to survive because they need to build proteins. One of the amino acids they need is tryptophan, they can find this in their environment or make it themselves using enzymes that are encoded by five genes. These five genes are located together in the gene and are called trp operon. These five genes are structural genes, controlled by a regulatory gene upstream.

Trp Operon - Repression - High Levels

When there are high levels of tryptophan in the environment, there is no need for the bacteria to waste energy on transcribing the structural genes. So, tryptophan binds to the repressor protein creating a conformational change, that allows the repressor to bind to the operator region. This prevents the transcription of the structural genes by blocking the path of the RNA polymerase.

Trp Operon - Repression - Low Levels

When there isn’t enough tryptophan in the environment, the tryptophan molecule that is binded to the repressor protein in the operator region will detach as it is needed. This removes the repressor, allowing the RNA polymerase to continue through to transcribing the structural proteins, generating more tryptophan until there is enough in the environment.

Trp Operon - Attenuation

Attenuation is a mechanism that aims to reduce the expression of the trp operon when levels of tryptophan are high. The difference between this a trp repression, is that rather than stopping the initiation of transcription, attenuation stops the completion of transcription, so once the RNA polymerase has passed the operator region.

Attenuation: Present/high levels of tryptophan (termination loop)

The ribosome runs past the tryptophan codons, stopping at the stop codon between domains 1 and 2, this stops domain 2 from binding with domain 3, so 3 binds with 4. This puts tension on the attenuator, so the mRNA strand pulls away from the DNA, so the RNA polymerase unbinds from the strand, ending transcription. Reducing tryptophan levels.

Attenuation: Absent (anti- termination)

The ribosome lags, stopping at two tryptophan codons waiting for tRNA to bring it tryptophan, due to low amounts of tryptophan. This lagging allows for 2 to pair with 3, preventing 3 from pairing with 4, creating a hairpin. Because this hairpin is further away from the attenuator, mRNA does not pull away and the RNA polymerase remains transcribing the strand. Creating more tryptophan.

Enzymes

Enzymes are molecules that act as catalysts to speed up biological reactions, most are proteins consisting of a tertiary structure to make them functional and in a 3D shape.

Active Site

The part of the enzyme to which the substrate binds.

Substrate

The reactant of a reaction that an enzyme catalyses. – the substrate and active site tend to be complementary in shape so that they can fit together, allowing the reaction to occur.

Features of Enzymes

They are reusable (as they aren’t consumed or broken down after one reaction), specific (often only bind to one specific substrate), reversible (enzyme catalysed reactions can be reversible with the same enzyme capable of building up larger molecules (anabolic) or breaking them into smaller ones (catabolic)), have an active site, are a protein, and they speed up not create reactions.

Lock and Key vs Induced Fit Model - Lock and Key

The substrate has a complementary shape to the active site

Lock and Key vs Induced Fit Model - Induced Fit

Originally not an exact complementary shape, with the bonds that form between the enzyme and substrate changing the sample of the enzyme so that the substrate fits.

Enzymes: Activation Energy

Activation energy is defined as the minimum amount of energy required to energise atoms of molecules to a state where they can undergo a chemical transformation. Enzyme speed up chemical reactions by lowering this activation energy.

Rate of enzymatic energy

All enzymes have specific conditions in which they function best, with the rate of reactions being impacted by temperature, pH level, and enzyme/substrate concentration.

Denaturation of Enzymes

The disruption of a molecule’s structure by an external factor such as heat

Effect of temperature on enzymes

Low - Little enzyme activity, because molecules collide less frequently

Optimal - The optimal temperature varies depending on the enzyme, but it is where the enzyme activity will peak.

High - Increased speed and motion of both enzymes and substrates, increasing enzyme activity. Because the higher kinetic energy results in more frequent collisions.

Too high- When temperatures reach levels higher than the optimal, the enzyme stability will decrease with thermal energy disrupting the hydrogen bonds. Causing the active site to lose its shape, resulting in a loss of activity – denaturation.

pH and Effect of pH

pH - Is the scale of acidity or alkalinity, with a low pH being a number less than 7, and a high pH being a number higher than 7 making it basic.

Effect - pH effects enzymes like temperature does, with there being a optimal pH level that allows for peak enzyme activity, with this optimal again differing depending on the enzyme. However, unlike temperature, the denaturation of an enzyme occurs if it is exposed to an environment that is higher or lower than the optimal pH.

Effect of substrate and enzyme concentration

If the enzyme concentration remains constant, while the substrate concentration increases then the reaction rate will also increase. But as this continues a point will be reached where all active sites will be occupied, so the reaction rate will no longer increase, and plateau, this is the saturation point.

Competitive vs Non-competitive Inhibition - Competitive

Where an inhibitor binds to and occupies the active site, blocking the substrate from binding, therefore no reaction occurs.

Competitive vs Non-competitive Inhibition - Non-Competitive

Where an inhibitor binds to a site other than the active site, causing a conformational change in the active site. This changes the shape, preventing the substrate from binding.

Reversible vs Irreversible Inhibition - Reversible

Where the inhibitor that binds with the active site forms weak bonds, so that they can be broken. So, the effects aren’t permanent and can be reversed and allow the substrate to bind. Slowing but not stopping the reaction rate.

Reversible vs Irreversible Inhibition - Irreversible

Where the inhibitor binds to the active site and forms strong bonds, so the binding is irreversible. So no matter how high a substrate concentration there is, no reaction will occur.

Enzymes that manipulate DNA

Endonucleases, ligases, polymerases

Endonucleases

A broad range of enzymes responsible for cutting strands of DNA. They do this by cleaving the phosphodiester bond of the sugar- phosphate backbone that holds the nucleotides together.

Ligases

Enzymes that join two fragments of DNA or RNA. They do this by catalysing the formation of phosphodiester bonds between the two fragments to merge them together. Essentially, they do the opposite of endonucleases, except they lack the specificity of restriction enzymes, as they can join any blunt or sticky end.

Polymerases

Synthesise polymer chains from monomer building blocks, with the main two types being DNA and RNA polymerases. These require a primer to attach to the start of a template strand of DNA. Primers are single stranded short chains of nucleotides that are complementary to the template strand. Once the polymerase attaches to the primer, it can synthesise a complementary stand to the template strand in a 5’ -3’ direction.

Recombination and Transformation - Recombination plasmid

A plasmid that has been edited to incorporate a target gene of interest.

Recombination and Transformation - Bacterial transformation

The process of the bacteria taking the recombinant plasmid from the environment.

Recombination Stage

1) The gene of interest is generated. 2) The plasmid vector is selected. 3) Restriction enzymes cut both the plasmid and the gene of interest to create complementary sticky ends. 4) Ligase joins plasmid and gene of interest by forming phosphodiester bonds in the sugar backbone.

Plasmid Vectors

Contain a restriction enzyme site (where the gene of interest is inserted), an antibiotic resistance gene, a reporter gene, and an origin of replication (ORI).

Transformation Stage

To insert the recombinant plasmid, bacteria will undergo heat shock or electroporation to make the plasma membrane more permeable, to allow the plasmids to pass the phospholipid bilayer and enter the cytoplasm.