Molecular Biology

Question 1

What are the three domains of life?

Answer 1

• Prokaryote (Bacteria)
• Archaea (same similarities with both prokaryotes and eukaryotes)
• Eukaryote (animals and plant)

Question 2

What is the structure of a phospholipid?

Answer 2

A phosphislipoid bilayer is essential from life and cell function and structure. The molecule has a very hydrophilic head attached to a phosphate group with a hydrophobic glycerol tail. (Google photo)

Question 3

How does a prokaryotic cell differ from a eukaryote?

Answer 3

The prokaryotic cell has very different properties to eukaryote they have
- no nucleus instead the DNA is free floating but compacted by DNA binding proteins to form the nucleoid
- extrachromosomal elements called plasmids free floating
- flagellum which is a long tail used for mobility
- pili which are on the outside and are present for intercellular communication and DNA and protein exchange.
- a capsule (slime layer) as an outer wall

Question 4

What are the two types of bacteria?

Answer 4

The two types of bacteria are distinguished their cell wall
- Gram positive. Has a thick peptidoglycan layer that provides strength and structure and no outer lipid membrane meaning they are more susceptible to antibiotics.
- Gram Negative. Thin peptidoglycan layer sandwiched between a cytoplasmic membrane and an outer membrane. The outer lipid membrane creates an additional barrier making them more resistant to antibiotics.

Question 5

How to differentiate between bacteria types ?

Answer 5

Under a Gram stain
- Gram positive turns purple
- Gram negative turns red/pink

Question 6

What is the general structure of a eukaryotic cell?

Answer 6

• Well defined organelle separated by individual membranes allowing each organelle to thrive in an individual environment (temp, pH)
• complex tube of interconnecting membranes called the endoplasmic reticulum
• a lipid protein layer separating the inside (cytosol) to the outside of the cell
• the cytosol compromises of a fluid ‘soup’ of ion, organic molecule, proteins and nucleic acids holding the organelles in place.

Question 7

What is the structure of the nucleus?

Answer 7

• The nuclear is surrounding by an outer nuclear envelope comprised of the inner and outer membranes. The outer membrane is continue with the ER.
• Nuclear pores are holes in the membrane allowing for the controlled transport of molecules in and out
• DNA is located in the form of chromosomes (formed from chromatids)
• nucleolus

Question 8

What is the role of the nucleus?

Answer 8

• hold and protect DNA
• synthesis and processing of rRNAs required fro the assembly of ribosomes (small and large ribosomal unit). This takes place in the nucleolus.

Question 9

What is the structure and rile on the endoplasmic reticulum?

Answer 9

This is an extensive network of interconnecting membranes that extend from he outer nuclear membrane to the cell. membranes. their are two types
- rough ER covered in ribosomes that are responsible for protein synthesis
- smooth ER which synthesises and stores various other compounds needed fro cell function. Also creates transport vesicles too carry proteins to the Golgi.

Question 10

What are ribosomes and their role in the cell?

Answer 10

Ribosomes are large nucleoprotein complexes made up of two protein subunits bound together by rRNA. They are either are floating or bound to the rough ER, there primary role is protein synthesis. The larger subunits creates more proteins as contain more rRNA.

Question 11

What is the structure and role of the Golgi apparatus?

Answer 11

This is a network of flat smooth membrane, sis and vesicles. It is responsible for protein transport and folding, membrane synthesis and formation of lysosomes.

Question 12

What is the structure and function of lysosomes?

Answer 12

Lysosomes are surrounded by a singles membrane that maintains a pH of 5 and are rich in hydrolase enzymes. They are responsible for the digestion of external material or old organelle and intracellular material. The hydrolase enzyme break down protein, lipids, carbohydrates and nucleic acid along the side the highly acidic conditions.

Question 13

What is the role of the mitochondria?

Answer 13

The mitochondria is the site of ATP synthesis suppling the energy for all cellular activities. They also contain their own DNA (mtDNA) which is inherently maternal and contains information fro around 13 mitochondrial proteins and some RNAs.

Question 14

What is the structure of the mitochondria?

Answer 14

The internal matrix (mitosol) is surrounded by two membranes, the inner is convoluted to form cristae embedded in it is the enzyme ATP synthase. The outer membrane contains large pores (porins) that allow for the transport of large molecules however int he inner membrane is very selective and contains transport systems to control the transport of metabolites in and out of the mitosol.

Question 15

What is the cytoskeleton?

Answer 15

Eukaryotic cells have a cytoskeletal network of dynamic filaments that assemble and disassemble design on the cells need. They maintain and change the shape of the cell and mediate cellular movement, cell division and transport whiting the cell.

Filaments are microtubules (tubilin) which provide the ability to assembly and disassembly rapidly and microfilaments (actin) forming F filaments in response to ATP binding and hydrolysis.

Question 16

What is the central dogma?

Answer 16

This describes the unidirectional flow of genetic material within the cell. Essentially
- DNA
- transcription into RNA (mRNA) a working copy of DNA
- translation into proteins

Question 17

What is the nucleic acids?

Answer 17

This are complex molecule that store and transport genetic information. The two main types are DNA and RNA which differ by their sugar and base composition. They contain all the information needed for a primary sequence of proteins.

Question 18

What is the basic structure of the nucleic acid?

Answer 18

Each nucleic acid is made up of the building blocks called nucleotides which all contain
- a five carbon sugar (deoxyribose in DNA and ribose in RNA)
- A phosphate group
- a nitrogenous base

They join together with a 5’ to 3’ direction

Question 19

What is the bonding within the DNA molecule?

Answer 19

Nucleotides are joined together by phosphodiester bonds between the 5’ carbon on one nucleotide and the 3’ in the next creating a a phosphodiester backbone. These individual strand are joined by hydrogen bonds between complementary bases (GC and AT). The two strand them twist together into a double helix shape allowing the bases to line up.

Question 20

What is the bonding within RNA?

Answer 20

RNAs primary structure consist of a chain of nucleotides linked by phosodiester bonds also 5’ to 3’. Bases are G,A,U,C, based attached to the 1’.
They also have a secondary structure in which the chain can fold into a 3D shape (loops and hoops) crucial for their function and interactions between other molecules.

Question 21

What are proteins?

Answer 21

A specific sequence of amino acids connected via peptide bonds (primary structure). They adopt specific structures by fooding and associating with other proteins and molecules. They have a wide range of function.

Question 22

What is the basic structure of an amino acid?

Answer 22

There are 20 types of ammonia acid with the general structure containing an amide group attached to a carbon with and changeable R group attached and a carboxylic acid group attached to the central carbon. Join together by peptide bonds.

Question 23

What is the transcription?

Answer 23

This is essential the ‘copying’ of DNA into mRNA. It involves the unzipping and copying the DNA bases from the 3’ to 5’direction by RNA polymerase.
It is written as

d 3’ A G G T 5’
(d meaning DNA, 3’ the starting direction, the individual bases and then 5’ the ending point)

The complimentary RNA will thus read as

r 5’ U C C A 3’

Question 24

Why are ever gene in all the cell expressed at one time?

Answer 24

Some genes are expressed all the time as they are essential for the normal working of the body however some gens are cell specific and express in different cell types. There are controlled systems in place to regulate this (outside factors place key role)

Question 25

What are the key stages in transcription?

Answer 25

1. initiation 2. elongation 3. termination

Question 26

What is initiation (transcription)?

Answer 26

Initiation is the most important part which is controlled by a region of DNA that doesn't code for genes called the promoter. A protein bind here and allow for the RNA polymerase to attached to the genome. The RNA polymerase has two sides the CTD and NTD which bind to the DNA then moves from a closed form to an open form pulling the DNA apart . Allowing the nucleotide to enter and begin to stabilise and elongate. RNA and DNA with aling anti parallel to each other.

Question 27

What is elongation in transcription?

Answer 27

As the nucleotides begin to flood into the open DNA the RNA polymerase enters elongation mode in which it catalyses phosphodiester bonds between the neighbouring nucelotides forming the mRNA strand until the terminal signal is reached.

Question 28

How are mistakes fixed in transcription?

Answer 28

Mistake happen in elongation (1 in ever 100,000 bases). - RNA polymerase can detect these mistakes and corrects them by backtracking along the transcription removing nucleotides that had just been added. If the RNA polymerase gets stuck in the backtracking state it is cleared out by other proteins - In bacteria these are called GreA and GreB which coordinate a Mg2+ ion in the active site of the RNA polymerase causing it to hydrolyse and detached from the DNA chain. - Mfd is a transcription repair factor that translocates along the DNA and 'bumps' trapped RNA polymerases out of the way

Question 29

What is termination in transcription?

Answer 29

This is the process in which RNA polymerase releases the DNA template and new mRNA there are two ways - intrinsic termination. This is when then mRNA forms a stem loop structure followed by a string of U when the RNA polymerase hits it, it will detach. - p- dependent termination involves a protein called Rho which binds to a specific sequence in the mRNA (5') and moves along until it reaches the RNA polymerase and 'bumps' it of the RNA polymerase.

Question 30

What is the structure of prokaryotic RNA polymerase?

Answer 30

The core is made up of four protein subunits. Two larger subunits (B', B) and two smaller subunits next to them (a).

Question 31

How to prokaryote regulate DNA?

Answer 31

Sigma factors (specific proteins) regulate the activity of RNA polymerase by making them specific to different promoters regions.They do this by binding to the core RNA polymerase and determine the promotor specific binding on RNA p. e.g sigma70 control house keeping genes, sigma 28 needed for flagellum and chemotaxis and sigma54 needed for assimilation of nitrogen.

Question 32

How do prokaryotic transcription differ from eukaryote?

Answer 32

- In prokaryotes transcription occurs in the cytoplasm and is coupled with translation as ribosome can attached directly to mRNA and start translating the RNA into proteins. Whilst in eukaryotes it takes place in the nucleus and is thus separate from translation. - In prokaryote have a system where a transcription factor can make a a long mRNA chain which contains multiple genes with a stop codon causing ribosome to drop. This is an efficient way of making a group of related proteins for a specific mechanism (get the whole set)

Question 33

What are the key parts in eukaryotic cell transcription?

Answer 33

As the DNA is within the chromatin the process must be more sophisticated. - other transcription factor must bind to the promoter region other then RNA polymerase to imitate transcription - enhancers and silencers are used to regulate transcription - they have three different types of RNA polymerase for transcribing different classes of gene (prokaryotes only have the one). RNAP II (nuclear transcription), RNAP I (transcription of ribosomal RNA) and RNAP III (transcription of RNAs within the cell) - genes have exon (coding regions) and introns (non bonding regions)

Question 34

What is the overview of the more complex control factors of eukaryotic transcription?

Answer 34

- Transcription factors will bind to the DNA first (in prokaryotic they would bind to the RNA polymerase) they recognise specific sequences (e.g. TATA and CAAT) and other transcription factor (proteins) then bind to that transcription factor and allow the recruiting of the RNA polymerase II to the correct starting site and assist in unwinding the DNA and initiate the elongation mode, before detaching. This allows for more controlled over transcription as each protein is controlled by specific environments. - There are also distant sites that are recognised by a protein complex which causes it to fold, it then come into transcription as either stimulate/ stabilises the RNA polymerase to work (enhancer) or will de-stimulate (silencer). Allowing the increasing or decreasing of transcription of a specific protein. - DNA is packaged away in the nucleus again allowing for more control over transcription

Question 35

How does the packaging of DNA help with the control of transcription?

Answer 35

DNA helix are packaged by interacting with the proteins called histones which wrap the DNA into regions to compact onto chromatins which compact again into chromosomes. The histones form bead like structures with the DNA called nucleosomes. There are four types (H2A, H2B, H3, H4) two of each with the DNA form the bead in which the DNA is wrap around forming this tight structure. Making transcription hard. There are a varied of modifications in various positions that can take place to a histone - phosphorylation - acetylation - ubiquitination (small protein) - methylation These all affect the stability of the nucleosomes or can form binding site in which other proteins can bind to again altering that stability.

Question 36

What is acetylation and how does it affect transcription?

Answer 36

This is the transfer of acetyl groups from acetyl CoA to histones (N terminal region of H4 and H3). This causes the histone to release the DNA slightly allowing other re modelling proteins to kick the histones out of the way facilitating transcription.

Question 37

What is methylation and how doe sit affect transcription?

Answer 37

Histone methyltranferases (HMTs) find a side chain on H3 and H4 histone that contains a lysines group and bind a methyl group to the amine group on the lysine. the nitrogen donate the lone pairs to the methyl group on a S adenosyl methionine (a methyl carrier) which detached methylating the lysine all catalysed by the HMTs. Repressor proteins (HP1) looks for these methylated group and when finds it promotes the binding of more methyl groups forming closed DNA (all histones clumps together into a heterochromatin) and prevent DNA being accessible for transcription.

Question 38

What is chromatin remodelling and how does in help control transcription?

Answer 38

Chromosomes can be managed by chromatin remodelling proteins which are very large protein complexes which are able to maintain DNA by monitoring the packaging and locate histone which need to be moved. It can loosen in, remove or shuffle thump and down DNA. (combing through the DNA) disassembling and reassembling histones using ATP.B

Question 39

What are the key difference between the eukaryotic and prokaryotic transcription? (simple bullet point overview)

Answer 39

- Translation factors bid to specific promote sites in eukaryotic but in prokaryotic they bind directly to the RNA polymerase - Eukaryotic use silencers and enhancer whereas prokaryotic do not - eukaryotic have three different RNA polymerases (I, II,III) versus only 1 in prokaryotic - more complicated uses of chromatin structures through interaction and modification of histones.

Question 40

How does a polypeptide chain form and why are they so strong?

Answer 40

Two amino acid form a polypeptide chain by a condensation reaction joininng the carboxylic group of one amino acid to the amide group of the other. They are very strong due to their resonance and planar structure. Resonance arises from the lone pair the nitrogen being drawn toward the oxygen creating a double bond character C-N bond. Some flexibility but can only twist around the phi and shi angles (the connecting peptide bonds between amino acids)

Question 41

What is the protein structure?

Answer 41

A protein is made up of a chain of amino acids (primary), the sequence of acids and their stereochemistry affect interaction between side chains and thus the shape of the protein. Two main ones are an alpha helix and beta sheet (secondary) held together by intermoicteuar interactions and hydrogen bonds. Tertiary structure can form when covalent bonds between R groups form giving a globular shape. This can then turn into a quaternary structure when two or more polypeptides can join to form a single protein (coding for this final structure found in the initial primary sequence).

Question 42

What is the genetic code?

Answer 42

This is the code that refer to the relationship between codon and amino acid, it is universal and used in every most organisms. A codon is a triplet of nucleotides (e.g. A U U, A G T) that codes for one of the 20 amino acids to be added to the polypeptide chain, additionally the start and stop codons. An anticodon is the

Question 43

What is a written example of how the genetic code works and related to an amino acid?

Answer 43

DNA 3'- d(CCG GCA CGT ...) - 5' complementary mRNA 5' - R(GGC CGU GCA ...) - 3' polypeptide chain H2N - (Ala - Arg- Ala ) - CO2H

Question 44

What are the key features of the rRNA on ribosomes?

Answer 44

The small and larger subunits that are comprised of multiple proteins and rRNA. The role of the rRNA acts as a recognition storage and has catalytic properties.

Question 45

What is tRNA and how does the amino acid attach?

Answer 45

This is another RNA used in translation which is called transfer RNA. Its structure is curled into an almost clover shape with hoops and loops. On the end it has an anticodon which changes per molecule and pairs with complementary codons on the mRNA. On the other end it has an arm region on the 3' end where the amino acid connect. tRNA matched with a protein using tRNA synthase which will use ATP and attach it to the amino acid making it an energetic leaving group. It then recognise the specific tRNA and attach the amino acid to the adenine creating the 'loaded' tRNA.

Question 46

What is the initiation of translation?

Answer 46

To begin translation it need to be initiated. initiation factors (proteins, depicted as hexagons) will use GDP (similar to ATP) to bind together a small ribosomal unit to a mRNA. A specific initiator tRNA (carries methionine in eukaryotes, formylmethionze in prokaryotes) will then bind to the start codon on the mRNA. A larger ribosomal subunit then joins the complex forming a functional ribosome. The start codon with the attached complimentary tRNA is the p site and the larger ribosomal subunit will attach in the A site before shuffling along to the p site finishing initiation.

Question 47

What is the processes of elongation in translation?

Answer 47

This is where ribosomes moves along the mRNA Elogantion factors will locate a loaded tRNA and will bring them to the ribosome. Only the correct tRNA will be able to enter the vacant A site and recognise the codon. The two amino acids are the connected together by the ribosome (lone pair on the amide group in the A group attacks the carboxylic acid group on the p site breaking the bond that held the initial amino acid to the tRNA). The initial tRNA detach and an elongation factor-2 protein then pulls the mRNA along the ribosome into the p site and the A site in then vacant again meaning it can repeat the process.

Question 48

What is the termination step of translation?

Answer 48

Termination factors stop the whole process. There is no tRNA for a stop codon however their is a protein called a release factor which come in to the A site and recognise the stop condon, it will then hydrolysis the bond between the tRNA and polypeptide chain. It will then kick the tRNA, splits the ribosome and kicks the mRNA off allowing the cycle to repeat.

Question 49

Why is protein synthesis so important in drug uses?

Answer 49

The whole process is very energy intensive using high amount of GDP and ATP making it useful for target drug molecules. As if protein synthesis is interfered with can shut down the whole bacterial unit.

Question 50

What are some key examples of drug targeting protein synthesis?

Answer 50

Streptomycin > binds to the small ribosomal subunit and alters its interaction with tRNA and mRNA resulting in misreading Neomycins/gentamicins > binds to the small ribosomal subunit and inhibit the translation process Tetracylines > bind to the small ribosomal subunit and interfere with amino acyl-tRNA binding Chloramphenicol > inhibits peptides transferasre by binding the active sire erythromycin > binds to the large ribosomal unit and interferes with translocation (ribosome moving along the mRNA) puromycin > resembles tRNA and binds to the A site an act as an acceptor of the peptides.

Question 51

How do bacteria target eukaryote protein synthesis?

Answer 51

Shiga toxins > These toxins makes an enzyme that targets RNA on the large ribosome. It cleaves an adenine base from the RNA blocking protein synthesis. Ricin > An N-glycosylase that cleaves an adenine residue on large ribosome subunit A-sarcin > cleaves rRNA of the large subunit Colicin E3 > cleaves RNA of small ribosomal subunit near the mRNA binding site

Question 52

What is the diphtheria toxin?

Answer 52

This is a bacterial toxin produces by corynebacterium diphtheria. The toxins is made up of two proteins one on each side the N terminal domain contains the catalytic domain and B side containing a transmembrane (T) domain and receptor (R) domain. The Receptor domain is recognised as a protein and the cell allows it to enter the cell through exocytosis. It then forms a lysosome around the protein in an attempt to breakdown the protein, however the acidic conditions causes it to spilt and the t domain binds to the membrane and forms a hole popping the lysosomes releasing the contents including the c domain. The c domain catalyses the ADP- ribosylation and thus inactives the Elongation factor II, will keep adding and adding which it what makes so toxic.

Question 53

What are the levinthal paradox?

Answer 53

This is the idea that proteins must fold via a pathway in which all random configuration are tested to find the one with the lowest energy (the native fold). This however would take a very long time and thus doesn't explain how protein fold in just milliseconds.

Question 54

How to proteins fold so quickly?

Answer 54

They fold primary according to the type of amino acids in the chain normally either in alpha helix or beta pleated sheets. The amino acids will interacting locally forming larger molecules then will fold, this will start the sequence by providing a framework of sorts for more foldons to form. A random structure may form that could be very stable this makes the misfolded protein kinetically trapped.

Question 55

What are molecular chaperones?

Answer 55

Many proteins fold to their native conformations naturally however some require the assistance of molecular chaperones. These help stabilise folding intermediates, maintain intermediates through the membrane, helps unfold and refold misfoled segments, prevent the formation of misfiled intermediates, prevent aggregation and inappropriate interaction either other proteins. They do this by binding to the hydrophobic regions of the proteins stabilising (pulling away for misfolding). They dont really happen in prokaryotic cells (only sometimes in archea)

Question 56

How are proteins transported and targeting?

Answer 56

This happen as they have a signal on the N terminal end that indicates their destination. A signal recognition particle will recognise and attach to the protein signal, stopping any translation. This complex will then bump into a receptor on the endoplasmic reticulum and bind to, it travels through the membrane and bumps into the translocon molecule and attaches the ribosome to it. The SRP (signal recognition particle) is then released and ribosome will then finish translation. The newly created protein then travel through the now open translocon pathway into the inside of the ER.

Question 57

What are the two end of the protein?

Answer 57

Proteins are always made from the N terminal end (the start) and finish at the c terminal.

Question 58

How does proteins enter the mitochondria?

Answer 58

Mitochondrial proteins are synthesised by the ribosomes in the cytosol. N terminal pre sequences mark proteins for mitochondrial import, this is a very positively charged alpha helix that can be recognised by a mitochondrial receptor, its transported through translocator (molecular charpeone) where it is unfolded and then threads it through the membrane into s the mitrochondria to be refolded, and the tag is then removed completely allowing it to stay inside th mitochondria or partial cleaved allowing it to be embedded in the membrane.

Question 59

How are proteins imported into the nucleus?

Answer 59

Again the protein is marked with a tag on the N terminal of the protein this is called the nuclear localisation signal a cluster of basic amino acid sequences. This act with carrier proteins (importins) that transport them through the nuclear pores. they are then released from the carried proteins though GTP hydrolysis allowing the carrier protein to return to the cytosol. Driven by GTP.

Question 60

What are the different post translational modifications?

Answer 60

There are multiple ways that a protein cane modified such as certain cleavages creating different proteins, fatty molecular are tagged a thus. Main ones are - methylation (adding a methyl group) e.g. histones - phosphorylation (ATP exchanges an OH for a phosphate group) - acetylation e.g. histones - hydroxylation (adding a -OH) - proline - sulfation - glycosylation

Question 61

What is the process of sulfation in post translation modification?

Answer 61

These are enzymes that manipulate sulphates. These enzymes are activated through a cysteine residue is converted to a C alpha formylglycine by another enzyme. This is then able to pick up a a sulphate group from metabolites. If you have a disorder which blocks the formation of formylglycine then all the sulpatases will stop working it will lead to a lack of metabolism and thus death.

Question 62

What is the process of n linked glycosylation in post translation modifications?

Answer 62

This is the attachment of an oligosaccharide (sugars) to a protein through the amide nitrogen of asparagine (Asn). A dolichol phosphate is tagged with these sugars (Glucose Glc) to begin and further sugars are added, then man sugar are added in a branching pattern (unique shape). Sugars are then flipped into the inside on the ER and more sugars are then added. This results is very specifically shaped glycan sugar inside the membrane, a protein with a specific amino acids sequence. It will be recognised by the glycol and the sugar will be transferred onto the protein. This will affect the folding pathway (thus must be added to allow them to fold effectively), and effects is transportation. if folded correctly in the ER the glucose sugars are removed, its then moved into he Golgi and modified some more with more sugars leaving a phosphorylated mannish residues which will then bind to the mannose 6-phosphate receptorwhcih is embedded in the golgi and will form a lysosome which is then transported to the membrane.

Question 63

What happens if n linked glycosylation cannot take place?

Answer 63

A genetic disorder can cause the inability of the protein to transfer the phosphate group onto the mangos meaning the GlcNAc-P glycotranferase (proteins ar enot tagged efficientlysnf thsu targeted) cannot function and thus no lysomes. This will lead to a a build up of proteins and enzymes with no destination (won't get into the lysosomes), this will show up as abnormal inclusion bodies (l cell disease).

Question 64

What is the O-linked glycosylation pathway?

Answer 64

This involves oligosaccharides attached to proteins through hydroxyl groups e.g serine (Ser) or Threonine (Thr). It happens once the folded protein has reached the golgi. Enzymes detect the -OH group at attached the N-acetyl glucosamine via an O link. No need for a specific amino acid sequences, the amount of enzyme available determine rate of glycosylation.

Question 65

What is protein degradation?

Answer 65

Proteins have varied life time, once no longer needed or damaged are removed through the proteasome. This is 28 protein that have come together, a protein will enter though a v capped complex that unfolds the proteins and breaks them down. The degraded fragments are then released from the opposite end. Ubiquitin (v small protein) is picked up by E1 in the cell and forms a thioester bond and then passes it to E2. E2 then passes it to E3 attached to a target protein it is then able to add the ubiquitin onto the protein using a lysine it can be chained over time. Enough tag will form for it to be recognised as a protein ready for degradation and picked up by the proteasome (only found in prokaryotes)

Question 66

What is the N - end rule?

Answer 66

This sates that the N terminal amino acids determines its stability/ half life on the protein.