Biology All Units

what is a protein?

one or many amino acid chains (polypeptides) folded into a 3D shape

what are the four structures of amino acids?

-central carbon atom bonded to a hydrogen atom

-a carboxyl group

-an amino group

-a variable r-group (each amino acid has a different r-group, which have specfic chemical properties that can affect how different amino acids interact with eachother)

what is a monomer?

the smallest building block of a polymer

what is a polymer?

a large molecule made up of small repeated monomer subunits

how do amino acids join together to form a polypeptide chain?

a condensation reaction- forms a peptide bond between adjacent amino acids

what are the four levels of protein structure

1. primary (the sequence of amino acids in a polypeptide chain)

2. secondary (when a polypeptide chain folds and coils by forming hydrogen bonds between different amino acids. it can form structures such as alpha helices, beta pleated sheets or random coils (irregular portions of secondary structure that join an alpha helix or beta-pleated sheet)

3. tertiary (the overall functional 3D shape of a protein formed when the secondary structure further folds and r-groups and amino acids interact and bond. this is the minimum level of structure that a protein needs to function)

4. quaternary (a structure formed when two or more polypeptide chains with tertiary structures join together)

what is a nucleic acid?

a large polymer composed of monomers called nucleotides (DNA and RNA)

what is the structure of a nucleotide?

-phosphate group

-five-carbon sugar (1' attaches to nitrogenous base, 3' attaches to phosphate group of FOLLOWING nucleotide, 5' attaches to phosphate group of nucleotide)

-nitrogenous base (adenine, thymine (or uracil in RNA), guanine and cytosine)

what kind of bonds join together nucleotides

phosphodiester bonds

what are the three main kinds of RNA?

-messenger RNA (mRNA): responsible for carrying genetic information from nucleus to ribosomes for protein synthesis

-transfer RNA (tRNA): deliver specific amino acids to the ribosome after recognising specific nucleotide sequences on mRNA

-ribosomal RNA (rRNA): the main structural component of ribosomes -messenger RNA (mRNA): responsible for carrying genetic information from nucleus to ribosomes for protein synthesis

what are four differences between DNA an RNA?

-DNA is double stranded whereas RNA is single stranded

-DNA contains the nucleotide thymine whereas RNA contains uracil

-DNA is inherited whereas RNA is temporary

-DNA is long-term whereas RNA is short-lived

what are groups of three nucleotides called in RNA and DNA?

RNA: codon

DNA: triplet

what amino acid does the start codon (AUG) also code for?

methionine

what pairing of nucleotides has a weaker bond?

adenine and thymine

what are the three kinds of RNA?

• messenger RNA (mRNA): carries genetic information from nucleus to ribosomes for protein synthesis

• transfer RNA (trNA): delivers specific amino acids to the ribosome after recognising specific nucleotide sequences on mRNA

• ribosomal RNA (rRNA): serves as the main structural component of ribosomes in cells

what are the four properties of the genetic code?

-universal: nearly all living organisms have the same codons to code for specific amino acids

-unambiguous: each codon is only capable for coding for one specific amino acid

-degenerate: amino acids can be coded for by multiple different codons

-non-overlapping: each triplet or codon is read individually without overlapping from adjacent triplets/codons

what are the six main regions of genes?

-promoter: an upstream (5' end) binding site for RNA polymerase

-introns: regions of non-coding DNA that are transcribed but removed during RNA processing and NOT translated

-exons: regions of coding DNA which are both transcribed and translated into the final protein

-termination sequence: a sequence of DNA that signals for the end of transcription

-operator: the binding site for repressor proteins, which can inhibit gene expression (found in prokaryotes only)

-leader: the section of DNA just upstream of the coding region and downstream of the promoter and operator that plays a critical role in regulating gene expression (prokaryotes only)

what are the three stages of gene expression?

-transcription (the copying of DNA into pre-mRNA): in eukaryotes DNA is large and cannot leave the nucleus so in order to transport the codes for a protein around the cell, so mRNA must be created in order to leave the nucleus.

in prokaryotes, transcription happens in the cytoplasm and in eukaryotes it occurs in the nucleus

-RNA processing: following translation, the pre-RNA molecule must undergo RNA processing before being sent to the ribosomes for translation (only occurs in eukaryotic cells)

-translation: after RNA processing, the mRNA molecule is ready for translation where it will exit the nucleus and travel to a ribosome where translation occurs

what are the stages of transcription?

1. (initiation) RNA polymerase binds to the promoter region of the gene and 'unzips/unwinds' the DNA

2. (elongation) RNA polymerase runs along the template stand of DNA (in. 3' to 5' direction), reading the nucleotide sequence and using free-floating complimentary RNA nucleotides to build a complementary single stranded RNA molecule called pre-mRNA (now uracil intsead of thymine)

3. (termination) RNA polymerase reaches the termination sequence of the gene and detaches, releasing the pre-mRNA and allowing the DNA strands to reattatch

what are the stages of RNA processing?

-a 5' methyl-G cap and 3' poly-A tail are added to the pre-mRNA

-the introns are removed and the exons are spliced together (by a spliceosome)

what is alternative splicing?

the process of removing some exons during the splicing process to allow for one gene to produce multiple different mRNA strands and code for different proteins

what are the stages of translation?

1. (initiation): the 5' end of the mRNA binds to the ribosome and is read until a start codon is recognised, when a tRNA molecule with a complementary anticodon will bind to the start codon and deliver the amino acid methionine to the ribosome, which signals for the start of transcription

2. (elongation): the ribosome moves along the mRNA and each codon is bound with a complementary tRNA anticodon. then, the tRNA molecule will deliver specific amino acids to the ribosome, which through a condensation reaction will form a peptide bond with an adjacent amino acid, growing the amino acid chain

3. (termination): the ribosome reaches the stop codon on the mRNA molecule, signalling the end of translation

what is a structural gene?

a gene responsible for producing proteins involved in the structure or function of a cell

what is a regulatory gene?

a gene responsible for the production of repressor or activator proteins

what is gene regulation?

a way for cells to conserve energy through either inhibiting or activating gene expression so proteins are not produced when they are not required

what is a repressor protein?

a protein that inhibits or decreases the expression of structural genes

what is an activator protein?

a protein which initiates or increases the expression of structural genes

what is an operon

a group of structural genes in prokaryotes that share a common purpose grouped in a way that their function is managed by a single promoter and operator

how does repression respond to low levels of tryptophan in a cell?

1. when there are low levels of tryptophan in a cell, there will not be enough tryptophan molecules to bind to the repressor protein

2. this causes the repressor protein to become inactive and detach from the operator region of the operon, allowing RNA polymerase to transcribe the structural genes involved in tryptophan production

3. this leads to an increase of tryptophan in the cell

how does repression respond to high levels of tryptophan in a cell?

1. when there are high levels of tryptophan in a cell, the tryptophan will bind to a repressor protein, allowing for a conformational change to change it into its active form.

2. this repressor protein will then bind to the operator region of the trp operon, preventing transcription of the structural genes by blocking the path of RNA polymerase.

3. this leads to a decrease of tryptophan in the cell

how does the process of attenuation respond to high levels of tryptophan in a cell?

1. the processes of transcription and translation will begin to occur simultaneously

2. the ribosome involved in translation arrives at the attenuator sequence that codes for two tryptophan amino acids. since there IS tRNA bound tryptophan in the cell, it will travel to the ribosome and add to the protein being made

3. the mRNA molecule being read by the ribosome will fold into a terminator hairpin loop

4. this loop causes the mRNA molecule to seperate from the template DNA at the attenuator sequence

5. RNA polymerase detaches from the DNA, causing transcription to stop before any structural genes are transcribed, therefore no new tryptophan is synthesised

what is exocytosis?

a form of bulk transport where the contents of a vesicle are released from a cell.

what are the stages of excocytosis?

1. a vesicle containing secretory products (such as a protein) is transported to the plasma membrane

2. the membrane of the vesicle fuses with the plasma membrane

3. the secretory products are released from the cell into the extracellular environment

what are the organelles involved in the protein secretory pathway (in order)?

1. ribosome: ribosomes synthesise proteins by assembling polypeptide chains from amino acids by translating mRNA

2. rough endoplasmic reticulum (ER): proteins to be secreted are synthesised in a ribosome in the rough ER whose environment allows for the correct folding of polypeptide chains

3. transport vesicle: a transport vesicle containing the protein will bud off the rough ER and travel to the golgi apparatus

4. golgi apparatus: the golgi apparatus adds or removes chemical groups from the protein before it is packaged into secretory vessels for export

5: secretory vessel: secretory vessels containing proteins for export will bud of the golgi apparatus and release the protein into the extracellular environment through exocytosis

what is an enzyme?

organic catalysts that speed up (catalyse) chemical reactions that would usually take longer to occur

what is a substrate?

the reactant that is undergoing an enzyme-facilitated reaction

what are the ten features of enzymes?

1. reusable (when an enzyme catalyses a reaction it is not used up, broken down or turned into a product)

2. specific (enzymes only bind to one specific substrate)

3. reversible (most enzyme catalysed reaction are reversible, with the same enzyme being able to build up larger molecules or break them down into smaller ones)

4. speed up not create (enzymes catalyse but do not create chemical reactions)

5. have an active site (each enzyme has an active site, which is the area to which the substrate binds)

6. are proteins

7. are a subset of catalysts (all enzymes are catalysts but not all catalysts are enzymes)

8. act on entire biochemical pathways (enzymes frequently influence entire biochemical pathways by catalysing each step)

9. end in '-ase' (most enzymes end with the suffix '-ase')

10. above the arrow (enzymes are written above the reaction arrow in a chemical equation)

what is activation energy?

the amount of energy required to start a chemical reaction

what is an anabolic reaction?

when two or more smaller molecules combine to form a larger one

what is a catabolic reaciton?

when a large molecule breaks down into two or more smaller molecules

how does temperature affect the rate of chemical reactions?

-chemical reactions speed up as temperature increases as molecules have more kinetic energy and collide more often

-when temperature decreases, there is less kinetic energy and the molecules collide less often, slowing down the rate of chemical reactions

-however once the temperature goes past the optimal, there is a risk the enzyme will denature

what is a tolerence range?

the wider range of temperature (and other conditions) which enzymes can still function in, even if it is not optimally

what is denaturation?

an irreversible change where the bonds that create tertiary and quaternary structure are broken

-in an enzyme this causes a conformational change in the active site of an enzyme, causing the substrate to no linger fit in it

How does pH affect rate of chemical reaction?

enzymes have optimal pH's where they function best

-however if exposed to an environment either above or below the optimal pH, an enzyme can denature

how does substrate concentration impact the rate of reaction?

-if enzyme concentration remains constant while substrate concentration increases, then the reaction rate will increase

-however once there are so many substrate molecules that continuously occupy all enzyme active sites, an increase in substrate will no longer increase the active rate (this is know as the saturation point)

how does enzyme concentration impact the rate of reaction?

-an increase in enzyme concentration can increase the rate of reaction due to the large number of active sites for the substrate to bind to

-similar to substrate concentration however, a saturation point will be reached where the rate of reaction will not increase further

what is a limiting factor?

a factor that prevents in increase in reaction rate

what are the four types of inhibitors?

-competitive (inhibitors that bind to an enzymes active site, blocking it so that the substrate can no longer attach to it- must have a shape complementary to the active site)

-non-competitive (bind to enzyme at a site OTHER than the active site (allosteric site) an causes a conformational change in the active site, preventing the substrate from binding)

-reversible (inhibiters that bind to the enzyme with bond that are weak enough to be broken, meaning their effects are not permanent)

-irreversible (bind to an enzyme with strong bonds that cannot be broken, meaning their effects are permanent)

what is a coenzyme?

an organic cofactor that binds to the active site of an enzyme and donates energy or molecules to the enzyme catalysed reaction.

(example includes ATP losing a phosphate group when releasing energy for a reaction and turning into ADP)

what is an endonuclease?

an enzyme responsible for cutting strands of DNA or RNA

what is a restriction endonuclease?

an endonuclease that target specific recognition sites

how do endonucleases work?

the enzyme will cleave the phosphodiester bond of the sugar-phosphate backbone that holds DNA nucleotides together

what is a blunt end?

a straight cut created by an endonuclease that leaves no overhanging nucleotides

what is a sticky end?

a staggered cut by an endonuclease that leaves unpaired, overhanging nucleotides

what is a ligase?

an enzyme that join two fragments of DNA or RNA together (not specific to sequences of nucleotides and can join together any blunt or sticky ends)

how does a ligase work?

a ligase will catalyse the formation of phosphodiester bonds between two RNA or DNA fragments

what are the two kinds of ligases?

-DNA ligase (joins DNA)

-RNA ligase (joins RNA)

what is a polymerase?

an enzyme that synthesises polymer chains from monomers

what are the two kinds of polymerase in gene manipulation?

-RNA polymerase (uses RNA nucleotides to synthesise an RNA strand during gene transcription)

-DNA polymerase (uses DNA nucleotides to synthesise a DNA strand during DNA replication)

how do polymerases work?

once attached to the start of a template strand of DNA by a primer, polymerases will read and synthesise a complementary strand in a 5' to 3' direction

how does CRISPR- Cas 9 work in bacteria?

1. (exposure) the bacteriophage injects its DNA into the bacterium, which will identify this as a foreign substance.

Cas 1 and 2 enzymes will cut out a short section of this DNA (right before the PAM sequence) known as a protospacer, which can then be inserted into the bacterium's CRISPR gene and become a spacer

2. (expression) the CRIPSR spacers will be transcribed (along with half a palindromic repeat from each side of it) and converted into an RNA molecule called guide RNA (gRNA). this gRNA will then bind to Cas9 to form a CRISPR-Cas9 complex

3. (extermination) the CRISPR-Cas9 complex will scan the cell for invading bacteriophage DNA that is complementary to the spacer on the gRNA.

when it finds it, Cas9 will cleave the phosphate sugar backbone to inactivate the virus

when the viral DNA is cut, enzymes within the bacteria will automatically work to repair it and until a mutation that makes the viral gene non functional occurs, the gRNA will repeat the process of finding an cleaving the DNA until a mutation does occur

what is a PAM sequence?

a short sequence of nucleotides that Cas1 and Cas2 can consistently recognise and know to cut the protospacer from just before the PAM sequence in the bacteriophage DNA

what are two things genetic modifications can do to a human genome?

-amend deleterious mutations (a change in DNA that negatively affects an individual)

-introduce biologically advantageous alleles into someones genome

how does CRISPR-Cas9 work for gene editing?

1. scientists will create synthetic sgRNA (single stranded guide RNA) that has a complementary spacer to the target section of DNA they want to cut

2. a Cas9 enzyme with an appropriate PAM sequence is obtained

3. Cas9 and the synthetic sgRNA are added together in a mixture and bind together to create the CRISPR-Cas9 complex

4. this complex is injected into a specific cell (such as a zygote)

5. the Cas9 will find the target PAM sequence and check that the sgRNA will align with the DNA

6. the Cas9 will cut the specific sequence of DNA

7. the DNA will attempt to repair the blunt end that has just been cleaved by the Cas9

8. when repairing the DNA, the cell may introduce new nucleotides into the DNA at this site. scientists may inject particular nucleotides sequences into the cell with the hope they will litigate this gap

what is 'knocking out' a gene?

knocking out a gene refers to the process of gene editing where scientists will prevent the expression of a target gene

what are the ethical issues with CRISPR-Cas9?

• in order to successfully alter ah organisms genome, a scientist must treat an embryo prior to the cells differentiating as this ensures all cells are altered (which means that they must experiment on embryos which is currently illegal and unethical)

what are the limitations of CRISPR- Cas9?

• in animal trials there has been success in eliminating genetic diseases, but this is yet to be the case in human trials

• in order to ‘knock in’ a new segment of DNA, scientists must introduce the nucleotide sequence they wish to add and hope it is taken up (process is imprecise and not consistently successful)

what is the polymerase chain reaction (PCR)?

a DNA manipulation technique that amplifies (duplicates) DNA

what are the four materials required for PCR?

-DNA sample

-Taq polymerase (a type of DNA polymerase obtained through bacteria, required instead of from human cells as it will not denature during the process of heating and denaturing the DNA sample)

-nucleotides

-sequence-specific DNA primers

what is the process of PCR?

1. denaturation (DNA is heated to approximately 90-95 degrees in order to break the hydrogen bonds betwee nthe bases and seperate the stands, forming single stranded DNA)

2. annealing (the single-stranded DNA is cooled to approximately 50-55 degrees to allow for the primers to bind to complementary sequences on the DNA)

3. elongation (the DNA is heated back up to 72 degrees in order for Taq polymerase to function optimally. It will bind to the primers on the DNA and synthesise a new complementary strand of DNA)

4 steps 1-3 are repeated until there is enough copies of the DNA

what are the two types of primers used in PCR?

-forward primers (primers that bind to the 3' end of the template strand, causing Taq polymerase to synthesise a new strand of DNA in the same direction as RNA polymerase would)

-reverse primers (primers that bind to the 3' end of the coding strand, causing Taq polymerase to synthesise a new strand of DNA the reverse direction RNA polymerase would)

what is gel electrophoresis?

a lab technique used by scientist to measure the size of DNA fragments

what is the process of gel electrophoresis?

1. DNA samples are placed in well at one end of the agarose gel with a standard ladder of DNA fragments with well-known sizes to be loaded in one well in order to estimate sizes of any unknown DNA fragments.

2. an electric current is passed through the gel (using one positive and one negative electrode)

the negative electrode is one the same end as the wells, with the positive one on the opposite side

as DNA is negatively charged it will move towards the positive electrode when an electrical current is applied

3. after a few hours the electrical current will be switched off and the DNA fragments will settle into bands

smaller DNA fragments will move faster than larger ones and so will therefore travel further than larger ones.

this means the DNA has now been seperated by size

4. the gel is stained with a fluorescent dye to allow for the bands of DNA to be visualised under a UV lamp

what is agarose gel?

a sponge-like jelly filled with tiny pores to allow for the movement of DNA fragments that is immersed in a buffer solution to help carry an electric current

how can you interpret DNA gels?

-the size of a DNA fragment can be determined based on it's position in the gel (smaller ones will be further from the wells than larger fragments)

-thicker bands indicates a greater number of DNA in that band

why are standard ladders needed in gel electrophoresis?

because DNA fragments of the same size will not always travel the same distance every time they are put in a gel.

this can be due to factors such as:

-voltage of the electrodes

-gel composition

-buffer concentration

-time

why are two kinds of primers necessary for PCR?

because both the template and coding strand need to be synthesised by Taq polymerase and the 5’ end of the coding and template strand are different

what are the four things required to create a recombinant plasmid?

• gene of interest: the DNA sequence of a particular protein wishing to be replicated will be isolated (using restriction endonucleases) and is amplified using PCR. this gene cannot have introns as bacteria do not undergo RNA processing to remove them

• a plasmid vector: a plasmid vector will be selected into which the gene of interest will be inserted

• restriction endonuclease: gene of interest and plasmid will be cut with the same restriction endonuclease to generate identical sticky ends (which will be complementary to each other to allow them to form hydrogen bonds)

• DNA ligase: added to join the gene of interest to the plasmid vector to create recombinant plasmid

what are the four requirements for a plasmid vector to contain?

• restriction endonuclease recognition sites (so plasmid can be cut and gene of interest can be inserted)

• antibiotic resistance genes

• an origin of replication - ORI (a sequence that signals the site of DNA replication in bacteria)

• reporter gene (a gene with a recognisable phenotype that can be used to identify whether a plasmid has taken up a gene of interest)

what are the two ways of inserting a recombinant plasmid into the cytoplasm of bacteria?

• heat shock

• electroporation

what are the steps for creating a recombinant plasmids for human insulin production?

1. plasmid vectors containing the amp^R and tet^R genes for antibiotic resistance (tet^R is reporter gene) are prepared

2. two plasmid vectors (one for each subunit of the insulin protein) are cut out with restriction endonucleases EcoRl and BamH1. DNA ligase then joins the subunit genes to the plasmids

3. plasmids are added to a solution of e. coli bacteria and some recombinant plasmids are taken up by bacteria

4. to determine which bacteria successfully took up the plasmids, they are spread and incubated on agar plates containing ampicillin. if they survive, they have taken up a plasmid of some kind.

5. to determine whether or not they took up a recombinant plasmid, the surviving colonies are spread on agar plates containing the antibiotic tetracycline (if they do not survive they must have the recombinant plasmid with interrupted tet^R gene)

6. the plasmids shown to have the recombinant plasmid are then cut again by EcoRl to insert another gene called lacZ (which produces B-glactosidase, a large enzyme, which will stop the smaller insulin protein being destroyed by digestive enzymes in e.coli)

7. recombinant plasmids containing lacZ were added to new solution of bacteria to be taken up

8. to determine which bacteria took up the plasmids, they are grown on agar plates containing ampicillin and X-gal. since B-galactosidase converts X-gal into a blue-coloured product, the colonies which grow and glow blue have taken up the desired recombinant plasmid

9. the bacteria with the recombinant plasmid are placed into conditions to exponentially reproduce. their membrane is then broken down and the fusion protein (insulin and B-galactosidase) are extracted before the methionine at the start of the insulin is broken down and seperated from the B-galactosidase to be used

what is a transgenic organism?

a type of genetically modified organism that involves the organism having genes from a different species inserted into its genome

what is a cisgenic organism?

a genetically modified organism that has genes from the same species inserted into its genome

what are the three stages in producing transgenic plants for argriculture?

1. gene identification: a gene of interest from another species (that provides some sort of beneficial trait for the host organism) is identified and isolated

2. gene delivery (the gene of interest is delivered into the cells of the host organism by direct insertion or use of a bacterial plasmid)

3. gene expression (transformed cells is grown repeatedly using plant tissue cultures before being applied in the field for agriculture use

what are some of the reasons GMO’s would be used in agriculture?

• increased crop yield

• increase nutritional value

• improve ability of crops to grow in different conditions

• increased disease resistance

what are antigens?

molecules that triggers an immune response

what are self- antigens?

antigens located on the surface of cells that mark the cells of an organism as ‘self’ so the immune system will not attack them

what are the two kinds of important self-antigens in vertebrates?

-MHC Class I marker (expressed on all nucleated cells in the body)

-MHS Class II marker (found on specialised cells in the immune system)

what is a non-self antigen?

antigens that the immune system recognises as foreign or not belonging to the organism

if a non-self antigen is recognised, the immune system is activated and attempt to attack it

what is an example of a malfunction involving antigens?

when the immune system recognises self-antigens as non-self and attacks self-cells (autoimmune disease)

or when there is an overreaction of the immune system to the presence of a harmless, non pathogenic antigen known as an allergen (allergic reaction)

what are the four kinds of pathogen?

• cellular (have a cellular structure and are living organisms)

• non-cellular (do not have a cellular structure and are non-living)

• extracellular (found outside a cell e.g. bacteria)

• intracellular (found inside a cell e.g. viruses)

what are the four main kinds of cellular pathogens?

• bacteria

• fungi

• worms

• protozoa

what is bacteria (and how do they reproduce?)

a unicellular prokaryote that can infect almost any part of the body.

they cause disease through the production of toxins or enzymes that cause cell death or affect their function.

bacteria reproduce asexually through binary fission

what are fungi (and how do they reproduce?)

eukaryotic organisms that contain long branching filaments called hyphae.

fungi reproduce asexually and sexually through spore formation

what are worms (and how do they reproduce?)

multicellular invertebrate parasites.

reproduce sexually through a complex lifestyle

what are protazoa (and how do they reproduce?)

single-celled eukaryotes that can be either free living or parasitic.

can harm cells in many different ways such as inhibiting nucleic acid or protein synthesis and various stages of cellular respiration.

protozoa reproduce through both asexual and sexual reproduction

what are the two kinds of non-cellular pathogen?

• viruses

• prions

what ae viruses (and how do they reproduce?)

an infectious agent composed of genetic material -RNA or DNA- inside a protein coat (caspid).

viruses cause disease through the lysis of cells during viral replication, the formation of cancer through affecting gene expression and overstimulation of the immune system leading to organ damage.

viruses cannot independently reproduce and instead insert their genetic material into a host’s cell and use the machinery of that cell to replicate

what are prions (and how do they reproduce?)

abnormally folded proteins that have the ability to induce normal proteins to become misfolded.

they only occur in mammals and affect only the brain and other neural structures

they are the only pathogen without nucleic acids and do not ‘reproduce’ but instead induce misfolding of nearby proteins to ‘spread’

what are the two parts of the immune system (in animals)?

-innate immune system (non specific immune responses)

-adaptive immune system (specific immune responses)