Nerves and Muscle - Week 5

Question 1

What is the amino acid pool?

Answer 1

• Organisms have an amino acid pool that exists within cells or within organisms that is distinct from the environment.
• This amino acid pool is used to synthesise proteins or muscle proteins in particular.
• The muscle protein synthesis is in equilibrium with the amino acid pool, and in a healthy adult, the rate of synthesis of muscle proteins equals the rate of degradation.

Question 2

What percentage of a male’s body weight is

muscle protein?

Answer 2

40-50%.

Question 3

What percentage of a female’s body weight is muscle protein?

Answer 3

30-40%.

Question 4

What are the mechanisms of muscle

catabolism?

Answer 4

• Catabolism/ muscle break down can occur in one of three ways.
• First by the ubiquitin-proteosome protein degrading system, a cellular level process where cellular proteins are tagged with ubiquitin and then targeted to the proteosome for degradation.
• This system works for lots of different host proteins including muscle proteins.
• Secondly, autophagy, meaning self-eating, where sub-cellular components, which could be organelles or proteins, are targeted to the lysosome for degradation.
• Finally, a role for calcium activated proteases or calpains, enzymes that break down proteins in a calcium dependent manner, such as titin, troponin and tropomyosin - but not actin or myosin.

Question 5

What is muscle breakdown and cachexia?

Answer 5

• Muscle breakdown occurs when breakdown exceeds synthesis and you get muscle atrophy, e.g. cachexia.
• This can occur in the late stages of cancer or HIV.
• Can also occur in response to release of cytokines such as TNFa, which cause increased degradation of muscle proteins.
• Also can occur due to increase in enzymes involved in ubiquitin-proteosome pathway if those are increased.
• Equilibrium will shift in other direction due to things such as starvation, that is the muscle proteins are degraded to replenish amino acid pool and there is a less muscle being synthesised as a result.

Question 6

What are essential and non-essential amino

acids?

Answer 6

-Mammals cannot synthesise all 20 naturally occurring amino acids.
-Therefore, must get some from diet.
-Essential amino acids which must be obtained from diet are nine amino acids, including histidine, methionine and valine.
-Non-essential of which are eleven can be synthesised within cell or inter-converted, so alanine, cysteine and serine.
-Now these are not amino acids you
need in diet as can be created from other amino acids or can be synthesised directly.
-To maintain this and to get all essential
amino acids need to maintain amino acid pool.
-Require 50g protein per day for a 70kg person, these are amino acids released by
proteins by digestion.

Question 7

How is the amino acid pool replenished?

Answer 7

• By ingested proteins which are digested by enzymes such as peptidase in digestive tract and then also by synthesis of non-essential amino acids, which contribute to amino acid pool.
• This amino acid pool as well as being replenished is being degraded over time and amino acids are degraded into three broad categories of waste products, ammonium (NH^4+), carbon skeletons and other breakdown products like purine, creatine, porphyrins and pyrimidines.
• These breakdown products of ammonium are eventually secreted from organism into environment, important for nitrogen balance.

Question 8

What is positive nitrogen balance?

Answer 8

-When nitrogen intake is greater than nitrogen loss, so you eat more nitrogen than excrete.
-Usually, a reason for that is a growing child and need to synthesise more muscle proteins than you would if you were in balance, pregnancy when need to synthesise
proteins for growing fetus, and tissue repair after injury, so when muscle proteins are damaged after injury or exercise, nitrogen
intake needs to be higher in order to repair tissue effectively and still maintain amino acid pool.

Question 9

What is negative nitrogen balance?

Answer 9

-Nitrogen intake less than nitrogen output, caused by inadequate intake of nitrogen or increased degradation of amino acids.
-So normal healthy adult, muscle mass is approximately 40-50% of weight, but examples of when you may be in negative nitrogen balance are starvation, illness, fever, ageing, as when you get to 75-80 years old, muscle proteins make up only 25% of body
weight, and also in cachexia, so could be in response to conditions such as cancer or other inflammatory conditions such as
COPD where muscle wasting occurs and therefore nitrogen intake is less than nitrogen output.
-So when in a state of negative nitrogen balance, the excreted products are greater than the ingested protein.
-This causes the equilibrium to shift around the system so that muscle proteins are more highly degraded to replenish the amino acid pool.
-Reason for this is that the amino acid pool is actually used to obtain carbon skeletons.
-So essentially muscles are in state of negative nitrogen balance, muscles are
considered non-essential so therefore muscle proteins degraded to replenish amino acid pool so therefore increasing number
of carbon skeletons can be generated from this pool by degradation of amino acids.

Question 10

How are amino acids converted to urea?

Answer 10

• The nitrogen groups are removed from the amino acids to form the ammonium ion.
• This ion is converted to urea via urea cycle.
• Ammonia exists primarily as ammonium, NH^4+, in solution, but ammonia, NH3, crosses membranes.

Question 11

How are amino acids transported from muscle to liver?

Answer 11

-Amino groups of amino acids in muscles and other tissues are transferred to other amino acids, either glutamate or alanine for
transport.
-In case of glutamate, the amino groups are then transferred to alanine or glutamine.
-Reason for this is alanine and glutamine are particularly stable and easier and safer to transport around body.
-So body protects itself by creating alanine and glutamine to safely transport nitrogen to liver.
-Once in liver, nitrogen flows from alanine and gluatmine to glutamate again, the
glutamate then releases the ammonium ion, and this ion converted into urea.
-As all of this happens in liver, liver therefore
considered gatekeeper of nitrogen balance as is responsible for flow of nitrogen out of system essentially.

Question 12

How is an ammonium ion formed?

Answer 12

In a two step process, so have transamination which happens twice, once in muscle to either alanine or glutamine, or in the liver to the glutamate, and then second step is oxidative deamination of glutamate to form the ammonium ion.

Question 13

What is transamination?

Answer 13

• Transfer of an amino group from alpha amino acid to alpha-ketoglutarate to form alpha-keto acid and glutamate.
• This is in equilibrium.
• So for example, in alanine, the alpha amino group, the amino group on the carbon skeleton backbone, is transferred to alpha-ketoglutarate to form glutamate, and also pyruvate, which is essentially deaminated alanine.
• This is catalysed by enzyme alanine aminotransferase and its cofactor is pyridoxal phosphate.
• Another example is asparate, so in presence of alpha-ketoglutarate, forms an equilibrium with oxaloacetate, which is deaminated asparate, and glutamate.
• Catalysed by enzyme aspartate aminotransferase.

Question 14

What is the oxidative deamination of

glutamate?

Answer 14

-Have glutamate and this oxidative deamination occurs in presence of NAD+ and water.
-In a reaction catalysed by glutamate
dehydrogenase, form ammonium ion, NADH and alpha-ketoglutarate as a product which is then recylced into the transamination
steps.
-Also, in equilibrium so can go either way.

Question 15

What is the urea cycle?

Answer 15

-Ammonium ion enters the urea cycle.
-In mitochondrial matrix of cell, ammonium in presence of a carbon donor, in this case it’s carbon dioxide, forms carbamoyl phosphate through activity of enzyme carbamoyl phosphate synthetase.
-Carbamoyl phosphate then combined
with ornithine to form citrulline, catalysed by activity of ornithine transcarbamoylase.
-Citrulline converted to arginosuccinate in
presence of aspartate via activity of enzyme arginosuccinate synthetase.
-Arginosuccinate converted to arginine through activity of enzyme arginosuccinase which also releases fumarate.
-Arginine then converted into ornithine through activity of arginase enzyme.
-This is the reaction in the presence of water that releases urea molecule and therefore synthesises urea through this cycle.
-It’s a cycle because urea can then be recycled back into the cycle to be combined with another molecule of carbamoyl phosphate to be continued round and round.

Question 16

What are carbon skeletons?

Answer 16

-Include things like pyruvate, alpha-ketoglutarate, fumurate, succinyl CoA, oxaloacetate.
-Synthesis of these is prioritised in case
of starvation.

Question 17

What are the concepts of glucogenic and

ketogenic amino acids?

Answer 17

-Glucogenic amino acids are those that contribute carbon skeletons to increase the concentration of glucose, so components of
TCA cycle for example or gluconeogenesis.
-Ketogenic amino acids contribute carbon skeleton that increase concentration of
ketone bodies, e.g AcCoA.
-Amino acids can be grouped into three group, either glucogenic, ketogenic or both.
-Two exclusively ketogenic, leucine and lysine.
-Large number of glucogenic amino acids like alanine and proline.
-There are some that are both like phenylalanine.”

Question 18

Why is classical conditioning applied in behaviour therapy?

Answer 18

-To help us understand how behaviours are learned, how behaviours are maintained and they can be modified or
changed.
-This is peritnent to behaviours that are problematic, or maladaptive, and need to be modified somehow so
they become adaptive and functional to an individual.
-These modifications happen by conditioning patterns of
emotional reactions and associated physiological responses.

Question 19

What is an experiment of classical conditioning?

Answer 19

-Experimental study of classical conditioning started by accident with physiologist Pavlov.
-He was not interested in
psychology, he was actually interested in function of dogs digestive systems.
-What happened was that during research, he noticed a weird phenomenon in dogs, that they’d sometimes salivate in response to stimuli other than food.
-So expected response is dogs would normally salivate when see food, and no other stimulus.
-This is what was later called Pavlovian conditioning, now classical conditioning.
-So it’s the type of learning that takes place when
behaviour becomes associated with the stimuli in the environment or triggers in environment through this
stimulus-repsonse association formed.
-Dogs started to salivate in response to other triggers or stimuli in environment which were presented to the dogs
just before food presented.
-For example, presence of lab assistant who would usually bring the food in, or sound of
bell that would signal feeding time.
- Wouldn’t expect dogs to salivate in response to these signals normally.
-So what happened here is that dogs learned a new behaviour.
-Their behaviour was conditioned by the environment.
-In this example they had developed an association between the bell sound and the food presentation.

Question 20

What are the three stages of classical conditioning?

Answer 20

• Before learning, during learning and after learning.
• Before learning or conditioning has taken place, the stimulus they are exposed to without conditioning is called the unconditioned stimulus.
• The automatic, natural and learned response to unconditioned stimulus is called the unconditional response.
• The new stimulus now, after being paired with the unconditioned stimulus, is called the conditioned stimulus as conditioning has taken place during learning.
• So what happens during learning, is that we are pairing another stimulus, a new stimulus, with the unconditioned stimulus, which is the original stimulus that caused the unconditioned response, and what trying to achieve is the conditioning that happens after learning has occurred, when new stimulus after being paired with unconditioned stimulus on a number of occasions will become conditioned stimulus and will elicit the conditioned response.
• So what would normally be the result of one stimulus, becomes that of another one due to associations that have been created.

Question 21

How do the stages of classical conditioning apply to Pavlov’s experiment?

Answer 21

-Before learning or conditioning has occurred, the food presentation will produce salivation.
-But the sound of a bell
doesn’t, so food presentation is the UCS and salivation is the UCR.
-During process of learning, the bell sound which would not originally produce salivation, is paired on numerous occasions with food presentation, so the bell sound
is presented and the food is presented straight after, and that’s the pairing.
-This process of paired presentation would
happen so many times until learning had taken place, and the dog had learned to associate the bell sound with the food, so the bell sound alone would produce salivation in the absence of food, and this is what would happen in the last stage, after conditioning has taken place.
-So the bell sound is now the CS, that would elicit CR of salivation which was not originally in response to that stimulus to begin with.

Question 22

What was the first empirical evidence of classical conditioning in humans?

Answer 22

-A series of experiments by Watson, performing a number of manipulations with an 11 month old baby, little Albert,
and he was able to demonstrate evidence of classical conditioning.
-Began by placing Albert on mattress in laboratory and rat would be placed near Albert.
-At this point he showed no fear of rat, he would play with it with curiosity.
-This is what would happen until presented Albert with sound of a bell which scared him.
-He cried and showed fear
as noise occurred.
-Would then repeatedly pair sound of bell with presentation of rat and after several pairings of two stimuli, when Albert would be presented with only the rat, he would cry and turn away from it, so would elicit a
fearful response which wasn’t there in the first place.
-Little albert had associated white rat which was original neutral stimulus with a loud noise, the unconditioned
stimulus, and ended up producing the fearful response of crying, originally the unconditioned response to noise, but now conditioned response to the rat.
-Watson performed other manipulations of this process and was able to show that the pairing of a stimulus with an unconditioned stimulus which would produce an unconditioned response
would result in that stimulus becoming the conditioned stimulus, leading to same response as unconditioned
stimulus, but now that response would be the conditioned response.

Question 23

What phenomena determine the strength of the conditioning that takes place?

Answer 23

• Temporal contiguity
• Generalisation
• Extinction

Question 24

What is temporal contiguity in terms of classical conditioning?

Answer 24

-The word temporal means time and contiguity means proximity.
-In general, shorter time the lapse is between the
conditioned stimulus and the unconditioned stimulus, the greater the strength of the association.
-The two stimulus closer in time, the greater the stimulus.
-So variations in temporal contiguity will affect the strength of the association.

Question 25

What is generalisation in terms of classical conditioning?

Answer 25

-After a conditioned response is acquired, stimuli that are similar, but not identical to the conditioned stimulus can also
elicit that same response.
-This is another phenomenon that determines conditioning and is called stimulus
generalisation.
-Usually, the greater the similarity, stronger conditioned response.
-Stimulus generalisation has some adaptive advantages for us, e.g.
-In terms of our survival, so it’s got some evolutionary purposes.
-Stimulus generalisation can also have disadvantages for example, most people would be frightened if shown a lion
in their garden, but this can them make them frightened by seeing a lion in a picture.

Question 26

What is extinction in terms of classical conditioning?

Answer 26

-When conditioning has extinguished, when unlearning takes place.
-Conditioned behaviours can become weaker until
they’re no longer produced.
-Extinction occurs when conditioned stimulus is repeatedly presented without the unconditioned stimulus.

Question 27

What is operant conditioning?

Answer 27

• As with classical, it is applied in behavioural therapy in order to understand how behaviours are learnt and maintained and how behaviours can be modified.
• However, the way this behaviour modification works in operant conditioning is different to that in classical.
• In operant, the focus is on increasing the frequency of existing adaptive behaviours and decreasing frequency of existing maladaptive behaviours.
• Also, on teaching new appropriate and adaptive behaviours.

Question 28

What is the Skinner Box?

Answer 28

-To study operant conditioning, Skinner devised the ‘skinner box’.
- In experimental manipulations with the skinner box, a rat would learn to press a bar in order to obtain food pellets from a cube.
- But it learned to do so under certain conditions, for example if rat pressed bar when a green light was flashed into the sample box, the food would be
released, so rat would be rewarded with food.
-However, rat would also learn after trial and error, that if it pressed the bar when the red light showed, there would be no food coming through but it would receive instead a mild electric shock coming through this shock grate embedded at the base.
-So rat would learn that it would operate on its environment in order to influence the consequences of behaviour.
-It could operate its environment by pressing on
the lever at certain points in order to influence the consequences of behaviour to get food and not electric shock.

Question 29

What is the A-B-C of operant conditioning?

Answer 29

-Skinner realised basic phenomena that show classical conditioning, such as temporal contiguity, stimulus
generalisation and extinction also apply to operant conditioning.
-In operant conditioning process, the basic components to remember are ABC contingencies.
-A stands for antecedent stimulus, which is the condition or situation in which the behaviour occurs prior to behaviour happening, so in our example it would be when the green light flashed into box.
-B stands for behaviour or operant, so in our example it’s the behavioural response when the rat presses the lever.
-C stands for consequence, what happens as a result of the operant behaviour, in our example it was when food was given.

Question 30

How is reinforcement a consequence of behaviour?

Answer 30

-So in operant conditioning process, the power of reinforcement is a vital element, as reinforcement as a process itself, will determine the consequences of behaviour.
-A reinforcer, a stimulus, a trigger increases the probability that an operant behaviour will occur again.
-There are two main types, positive and negative, and these refer to stimuli or
situations of any context that would be relevant to that operant behaviour.
-Positive reinforcement involves increasing
frequency of behaviour by following it with a presentation of a positive stimulus, a positive reinforcer, a pleasant
experience, a stimulus that would be desirable by individual.
-Positive reinforcement strengthens a response, if it’s
experienced after that response occurs.
-Negative reinforcement strengthens behaviour but involves increasing frequency of behaviour by following it with removal of a negative reinforcer.

Question 31

Why is positive punishment deemed to be less effective?

Answer 31

• May arouse negative emotions or aggression.

- Many not generalise to other situations/ behaviours and doesn’t teach desired behaviours.

Question 32

How does temporal contiguity determine the strength of the operant conditioning?

Answer 32

-In operant conditioning, stronger when delay in receiving a reinforcement is short.
-Immediate consequences of
behaviour affect the behaviours more strongly than delayed consequences.

Question 33

How does generalisation determine strength of of operant conditioning?

Answer 33

-Conditioned behaviour transfers to antecedent situations or stimuli that are similar to the original trained one.
-Occurs without further conditioning after generalisation of reward contingency. e.g. rat presses lever when seeing green light
(original conditioned stimulus) as well as when hearing a sound (conditioned stimulus after generalisation).

Question 34

How does extinction determine strength of operant conditioning?

Answer 34

• Gradual decrease in behaviour until it is no longer produced.
• Occurs when behaviour is no longer associated with its
  consequences. e.g. rat pressing lever no longer produces food.

Question 35

What is shaping?

Answer 35

• Reinforcement of successive approximations to the desired behaviour.
• Break desired behaviour into series of simpler steps.
• Start training simple behaviours and build up gradually to more complex ones.

Question 36

What are techniques adopted within classical conditioning?

Answer 36

-Re-conditioning and counter-conditioning.
-Re-conditioning involves relearning an association in order to lead to the
replacement of the maladaptive response with an adaptive alternative response.
-Counter-conditioning is when there is an alternative response, but one that is opposing the nature to the originally conditioned response.
-Type of exposure will usually be gradual, so in form of graded exposure, so is not full exposure as would see in flooding, which can be stressful to the individual, and is the least preferred in clinical practice.

Question 37

What is flooding?

Answer 37

-Based upon extinction.
-No gradual exposure, the person is placed in the most feared situation straight away.
-The least preferred option, and today is almost completely replaced by graduated exposure or systematic
desensitisation.

Question 38

What is systematic desensitisation?

Answer 38

-Based on gradual exposure and counter-conditioning.
-Entails training the patient in deep muscle relaxation,
constructing a hierarchy of anxiety-provoking situations, and presenting each situation seriatim to the imagination of
the person in a relaxed, non-anxious state.
-Owing to recent technological advancements, can be supplemented or replaced by real-life exposure, i.e. in vivo desensitisation.

Question 39

How does translation show similarities with transcription and replication?

Answer 39

-For one they are processes undertaken by large macromolecular machines which can contain proteins whose proper functioning seems to depend upon a variety of specific and nonspecific protein interactions.
-Secondly, these processes often involve accessory factors, so helpers that aid them at the different stages of the various processes, like initiation, elongation and termination.
-Although translation involves base pairing between complementary nucleotides, unlike the other polymeric processes we have, we’re not ending up with a nucleic acid product, this time it’s amino acids that get joined together to make the polymeric process.
-Thirdly, just like transcription and replication, translation is executed with accuracy.
-Finally, this polymeric
process, as with transcription and replication, is endergonic, it requires an input of energy from the cleavage of high energy phosphoanhydride bonds.

Question 40

How can a sequence made up of four nucleotides specify up to 20 amino acids?

Answer 40

-Can’t have a one-to-one coding ability between nucleotides and amino acids, there’s only four nucleotides within DNA, and if it was one-to-one correspondence with amino acids, that would leave 16 of your standard amino acids unaccounted for, they couldn’t be coded for.
-So need to start grouping these nucleotides together in order to code for more than four amino acids.
-This idea of bases being read as a bunch has been given the term a codon.
-A bunch of nucleotides and their bases is a codon
and will need several of them to specify a single amino acid.
-If had codons made of 2 bases, so a doublet code, that’ll allow you to code up to sixteen amino acids.
-Your variety of arrangements of nucleotides or doublets could give you 16 versions of these doublets, which would correspond to 16 amino acids, which would still leave us four short.
-So need a codon made of more than two bases.
-A triplet code allows a wide variety of arrangements that can be had – 64 triplet arrangements of the bases. -64 outstrips 20 standard amino acid types, so suggests it’s a triplet code that the genetic code is actually based on.
-This ability to have more than one codon specifying a particular amino acid, means the genetic code is degenerate.

Question 41

How are the bases in DNA and RNA grouped

into codons?

Answer 41

-Two potential options.
-Could be overlapping or could be sequential.
-If look at nucleotides within mRNA, if code was overlapping, say first codon is ABC, would code for first amino acid, and then second would be BCD, and third would be CDE etc.
-If code was non-overlapping, ABC would be first codon, DEF would be second codon etc.
-Turns out genetic code is a non-overlapping,
degenerative, triplet code.

Question 42

What does it mean if you have a suppressor mutation?

Answer 42

• If had suppressor mutation occur, say an insertion, just downstream of original deletion, depending on where mutations are, potentially this form of suppressor mutation, once it occurs, means you actually have a functional protein.
• Does job protein was intended to do.
• If add three nucleotides actually, can end up restoring original reading frame.

Question 43

How many active reading frames can any nucleic acid have?

Answer 43

Any nucleic acid may have three ‘active’ reading frames, therefore 1 polynucleotide could encode up to three polypeptides.

Question 44

What does the genetic code show?

Answer 44

-Genetic code highlights the idea that the code is highly degenerate, so have a redundancy in the code, for example leucine, has
six particular codons which can encode it.
-In some cases, the amino acids only encoded by one particular codon, e.g.
methionine.
-Most cases degenerate.
-Many synonyms generally only differ in position of final nucleotide within codon.
-Changes in the first codon position generally specify similar, if not necessarily the same amino acids.
-There are codons where can have second position where have pyrimidines changing between them, so have cytosines and
uracil’s.
-They generally give hydrophobic amino acids, whereas the second position purines changes between the guanines
and adenines.
-They tend to give polar amino acids.
-So this observation suggests a non-random origin to the genetic code and suggests it has an inbuilt ability to deflect the deleterious effect of mutations.
-Nonsense codons/ stop codons are UAA, UAG and
UGA.
-These are the kind of codons that never give a result, they’re not designed to ever bind a tRNA as they end up terminating polypeptide chains.

Question 45

What is another start codon, that is rare?

Answer 45

GUG.

Question 46

What is the structure of tRNA?

Answer 46

-We know that cells must translate the language of RNA base sequences into polypeptides, and have to have a translator to do
that.
-In 1955, Watson and Crick hypothesised had adaptor molecules and we now know those to be tRNAs.
-tRNAs tend to have
similar structures to one another.
-Tends to be between 54 to 100 nucleotides and if look at secondary structure have a cloverleaf structure.
-All of the tRNAs share some characteristics, have 5’ terminal phosphate group, a roughly seven base paired stem, known as the acceptor stem, the part of the tRNA which has the amino acid residue attached to it, attached to a 3’ terminal hydroxide group.
-At some parts of these acceptor stems, get non -Watson and Crick base pairing.
-Can see a three to four base pair stem ending in up to a seven nucleotide loop, this is the D-arm, because contains a non-Watson and Crick base pair called dihydrouridine.
-There’s a part of the tRNA which contains the anticodon, the anticodon loop.
-Range of post transcriptionally modified bases present.
-Don’t think these modifications are required to maintain the tRNA structure.
-Actually think they have a role in ensuring the correct amino acid is attached to the acceptor stem.
-Potentially, strengthen codon and anticodon interactions.
-Many biological macromolecules have a function that depends upon there structure and tRNA is no different.
-Has certain structural characteristics that ensures it does its job properly.
-Some of that is its shape in 3D space, so its tertiary structure, it’s quite a significant L shape.
-Different stems bind to one another.
-Important they’re as thin as possible, so up to 25 angstroms wide, which is fairly compact considering complexity of cloverleaf structure so several tRNAs can fit close together and the anticodon can meet the codons.

Question 47

What does the L shaped structure of tRNA mean?

Answer 47

That compact tertiary L shaped structure means many of the bases are protected against attack or interaction with outside
players for example, except the anticodon bases which need to bind with mRNA, but also part of acceptor stem as they have to accept the amino acids.

Question 48

What process loads the amino acids onto a tRNA?

Answer 48

-This is a process catalysed by aminoacyl-tRNA synthetases, the enzymes that load an amino acid onto the tRNA.
-In order for translation to work optimally, need to make sure this is an accurate process, and the correct amino acid is selected to be added
to the correct tRNA by this enzyme.
-This aminoacylation (amino acid addition to the tRNA) is going to be performed in two steps.
-First need the involvement of ATP and that has phosphate removed which then means can stick on an adenylate moiety onto your amino acid.
-Finally, this adenylated amino acid gets stuck onto your tRNA by your aminoacyl-tRNA synthetase. -By doing that releases adenosine monophosphate.
-What drives this reaction to completion, is the fact that you’re releasing this
pyrophosphate group, these two phosphates, this is a particularly high energy intermediate, and it will often go and get hydrolysed immediately by other reactions requiring an input of energy, so it draws reaction to right, leading to formation of aminoacyl-tRNA, so your tRNA containing your amino acid.

Question 49

What is aminoacyl-tRNA synthetase?

Answer 49

-This enzyme will be responsible for loading an amino acid onto more than one tRNA, as of course have degeneracy of genetic code, more than one codon leads to a certain amino acid being added to your polypeptide sequence.
-Going to need an enzyme
that recognises this class of tRNAs that will contain your amino acid of choice.
-There’s this subclass of tRNAs that all have the
same amino acid, they get called isoaccepting tRNAs.
-To recognise tRNA correctly, the synthetase will have a range of
interactions with the tRNA structure.
-Interacts with anticodon and acceptor stem to permit a recognition of tRNA, so aminoacyl-tRNA synthetase recognises correct tRNA before it goes looking for amino acid.

Question 50

What is proofreading?

Answer 50

-Aminoacyl-tRNA synthetases, as part of the charging process, have to select the correct amino acid to bind to the tRNA of choice.
-It is an accurate process.
-Most well-known example is the isoleucine aminoacyl-tRNA synthetase, that manages to select 40,000 isoleucine’s for addition to the tRNA for every valine that it accidently incorporates.
-Valine only differs to isoleucine by a single methyl group.
-So have a bit of a two-step process going on with proof reading.
-First of all, going to have to have correct amino acid being selected for and many of the active sites of the enzymes won’t allow amino acids bigger than the enzyme it’s evolved to work with to enter.
-Also have idea that aminoacyl-adenylates, the activate form of the amino acid, is then subjected
to another proof-reading step by the existence of a second active site within the enzyme.
-In this case, looking at isoleucine aminoacyl-tRNA synthetases which has two active sites.
-One activates the amino acid of choice and excludes all bigger amino
acids, but in this case concerned that maybe smaller amino acids e.g. valine have been activated.
-But then have proof reading step where the enzyme then tries to force the amino acid into a second site, and if that were to enter, which in case of valine it does, need to hydrolyse and remove it.

Question 51

What is the ‘wobble’?

Answer 51

-Know there are 64 codons potentially to be used within genetic code within eukaryotes, and three of those are nonsense codons so don’t code for an amino acid, giving 61 codons left.
-Don’t need 61 separate tRNAs as many of them are capable of recognising more than one, even up to three codons, as they show an ability known as wobble.
-Tend to have, especially in third codon anticodon position, so if looking at 5’ part of anticodon, often contain non-Watson or crick base present and those have ability to complementary base pair with more than one base, so can be a guanine that has a two prime methyl group added to
it, or an inosine.
-Inosine can complementary base pair with uracil, cytosine and adenine.
-So just by having one particular non-Watson Crick base in third anticodon position, suddenly gives you ability to bind three different codons.

Question 52

Why don’t you necessarily need 61 tRNAs?

Answer 52

-Due to wobble, don’t necessarily need your 61 tRNAs, in fact, many cells will have around 32 tRNAs or greater.
-Not to say all cells will go for minimal tRNA complements and rely on wobble.
-There is codon bias as there are frequently used codons within a particular organism.
-May only use three out of the six codons that code for leucine for example, it’s just evolved that way. In
which case, you’ll often find the tRNA species within those cells tend to be preferentially organised to bind with most frequently
used codons, meaning protein synthesis can occur more rapidly.