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MCD > Gene expression, protein synthesis and antibiotics > Flashcards

Gene expression, protein synthesis and antibiotics Flashcards

1
Q

The chemical structure of double-stranded DNA

what do the bases pair with?
what do they bind to?
what is the overall structure?

A

A only pairs with T
G only pairs with C

Phosphate deoxyribose backbone
2 polymer strands

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2
Q

DNA replication = semi-conservative

meaning?

A

Parent DNA double helix will break off and the new strands are formed by complementary base pairing

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3
Q

What are genes?

A

Genes are stretches of DNA that contain information for making RNA, mostly protein-encoding mRNA

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4
Q

What does transcription produce?

same as?

A

Transcription produces single-stranded RNA that is complementary to one strand of DNA

Will be the same as the ‘sense’ strand

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5
Q

sense and anti sense strands

A 
sense = 5 to 3 end
anti-sense = 3 to 5 end
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6
Q

structure of RNA (3)

A

RNA:
Single-stranded
Ribose instead of deoxyribose
Uracil instead of thymine

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7
Q

RNA in mammalian cells

what is majority of RNA in cells?
what is sedimentation coefficient?
how many components for rRNA?

different abundace of the 3 RNA in %

A

Majority of the RNA in cells is rRNA
Sedimentation coefficient is the speed at which ribosomes sediment in a centrifuge
rRNA has 5 different components
mRNA size is highly variable as it depends on the size of the protein

rRNA  = 80-85% cellular abundance (most common is 28S and 18S)
tRNA = 10-15%   (4S)
mRNA = 2-5% (highly variable)
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8
Q

Forms of rna (4)

what are they and their function?

A

rRNA: ribosome structure (RNA pol I)

mRNA: transcribed by ribosomes into proteins (RNA pol II)

tRNA: matches amino acids to codons specified by mRNA (RNA pol III)

snRNA: forms spliceosomes at splicing sites to splice out introns (RNA pol III)

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9
Q

mRNA Processing in Eucaryotes

what is mRNA?

what forms the primary RNA transcript?

what are the 3 transcriptional changes? explain them

A

mRNA carries the message from the DNA to make a polypeptide

• transcription of the DNA first forms the primary
RNA transcript

• the primary RNA transcript goes through post
transcriptional changes

5’ capping
- methylated guanine nucleotides put on
back to front at the 5’ end of the mRNA

polyadenylation
- poly A polymerase adds a whole series of A nucleotides at 3’ end of the mRNA

splicing

  • introns are sequences that do not code for proteins
  • these introns are removed by splicing (via spliceosomes)
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10
Q

Mature mRNA in Eucaryotes

after post-transcriptional changes, what is formed?

what are UTRs? what are they involved in? (2)

A

• after the above processes, the mRNA is formed
- only a small portion of the mRNA is the coding region

  • Untranslated regions are called UTRs
  • 5’ UTR is involved in the rate at which the message is translated to a protein
  • 3’ UTR is associated with stability of the mRNA
  • both of these UTRs determine how much protein is made
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11
Q

Importance of 5’UTR and 3’UTR

Role of UTRs (2)

what do they dtermines? (3)

A

If we look at the mRNA structure, we can see that the middle is the coding region, but on either side are untranslated regions (UTRs), so there is a 5’-UTR and a 3’-UTR.

These UTRs do not code for anything but are very important because for example in the case of the

5’ UTR it determines the rate at which the protein is synthesised and

the 3’ UTR tends to effect the stability of the RNA (how long it stays in cell before degrading).

So together, these UTR’s determine how much protein is made, as it determines the speed at which is made and the longevity of the message.

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12
Q

mRNA is Decoded in Sets of Three Nucleotides:
The Genetic Code

what is the first codon?
how many possible comninations? effect of this?

A

• the codon in the mRNA specifies an amino acid

  • a codon is a group of 3 nucleotides
  • the first codon on mRNA is always AUG forming methionine
  • there are 64 combinations possible, and so the genetic code is redundant to reduce errors
  • there are also various stop codons that stop translation
  • for some amino acids, a change in the 3rd base has no effect on the resulting amino acid
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13
Q

The genetic code is degenerate

total number of aa and total number of possible codons?
effect of this? why?

what are stop codons?

what does generality in genetic code mean?

A

As triple codons are used and there are 4 possible bases it means 4x4x4 combinations = 64 possible codons. However there are only 20 amino acids. This represents a type of redundancy, with more than one codon for the same amino acid (a degenerate code). This protects against the deleterious effects of mutations (so they result in silent mutations)

There are also STOP codons, which tell the ribosome to stop translating.

There is a great amount of generality in the genetic code (with few notable variations), which supports evolution. In other words this means, this genetic code is used by the majority of biological organisms.

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14
Q

Steps in protein synthesis (4)

what takes place?

A

Charging tRNAs with amino acids

Initiation of polypeptide synthesis (assembly of the ribosome on mRNA together with the first aminoacyl tRNA (met-tRNAi))

Elongation of the polypeptide (addition of amino acids one at a time)

Termination of polypeptide synthesis (release of the polypeptide)

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15
Q

Steps in protein synthesis
Charging tRNAs with amino acids

what gived tRNA a 3d structure?

components involved? (3)

what is broken down to provide energy for the reaction?

where does AA attach?what is this and what sequence is this? what group attaches here?

what detecst the specific tRNA and anticodon? effect of this?

A
  • there is intramolecular complementary base pairing within the tRNA giving it a 3D structure
  • components involved: ATP, amino acid, and tRNA
  • the PPi released is broken down by enzymes to Pi which provides energy to run the reaction
  • the amino acid attaches to the acceptor stem which is a CCA sequence in the tRNA
  • the carboxyl group attaches to the acceptor stem
  • aminoacyl tRNA detects the specific tRNA and anticodon and attaches the amino acid to the acceptor stem
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16
Q

Steps in protein synthesis
Charging tRNAs with amino acids (different explanation)

what enzyme is used to attach the tRNA to codon? what do you need for this reaction? what end does the attachment take place?

What ribsome is needed?
what are the 2 sub units? what happens when you have potein synthesis?

what are the 3 parts to the sub units?
describe their functions

A

The first is the attachment of the amino acid onto the tRNA, which is a crucial step. This is because it’s the tRNA that recognises the codon, and if the wrong amino acid attaches then the wrong amino acid will be delievered to the ribosome.

This step involves an enzyme called aminoacyl synthase

So the amino acid is attached to the tRNA by the aminoacyl tRNA synthase, which uses ATP to attach the amino acid onto the 3’ end of the tRNA. This process as stated earlier is crucial in getting the right protein. Note this is an energy dependent process and costs 1ATP per amino acid.

The tRNA is normally described as having a clover leaf structure, but in 3D structure it looks quite different.

The 80S Eukaryotic Ribosome:

This is the functional machine in translation, it is composed of two subunits, a large (60S) subunit and a small (40S) subunit. These assemble together on the initiation of protein synthesis.

There are three parts to the subunits, Exit, Peptide and Aminoacyl parts.

As the polypeptide is synthesised it gets extruded out of the peptide groove in the ribosome.

The aminoacyl groove is where the next amino acid comes in and sits.

The exit site is where the consumed tRNA (lost AA), leaves.

17
Q

Steps in protein synthesis
Initiation of polypeptide synthesis

what does this involves ther assembly of?

where does the mRNA and ribosome bind? what are the regions called? what are 2 key sites?

what happens at the P site of the ribosome? what does this then bind to and what does this act as? what does this allow and lead to?

what now assembles? and what is hydrolysed? what binds to the A site?

A

• this involves the assembly of 40S on the cap of the
mRNA

• the mRNA and ribosome bind at different regions of the mRNA

  • these regions are called initiation factors
  • there is a cap binding site
  • there is a polyA binding site
  • one more initiation factor binds to the initiator tRNA
    (Met tRNAi)
  • this happens at the P site of the ribsome
  • this then binds to GTP
  • GTP acts as a signaling component
  • this allows the small ribosomal sub unit to assemble at the capped end of the mRNA
    • the small ribosomal subunit then moves along the mRNA scanning for the AUG sequence
    (start sequence)
  • this uses a lot of energy
    • the large 60S subunit now assembles on top of the small subunit
  • hydrolysis of a GTP molecule now takes place
    • the 2nd aminoacyl tRNA now binds to the A site of the ribosome
18
Q

Steps in protein synthesis
Initiation of polypeptide synthesis (different explanatuon)

where do initiation factors assemble and what do they have bound to it? what do these bind to and what does that have bound to it? what are two types of these molecules?

what can only bind to the peptide binding site? where do the rest go?

where does the small subunits bind to? what must happen now and what does this require?

once this happens, what can take place? what does this result in? how does elongation take place?

A

This is where the ribosome assembles on the message. This involves initiation factors which recognise bits of the mRNA message. As the mRNA is translated it usually has a loop structure.

These initiation factors assemble on the small subunit and have a GTP bound to it, this binds to a special tRNA which has methionine bound to it. There are two types of methionine tRNAs. One which goes with the AUG to initiate translation and another which is involved in adding methionines in the body of the protein.

This initiation tRNA is the only one that can bind to the peptide binding site on the small ribosomal subunit. All the others, bind to the A (aminoacyl) site.

The small subunit binds to the 5’ cap end of the mRNA, the actual initiation codon may be much further down, so the ribosome needs to move along the message until it finds the initiating AUG. This uses ATP.

Once this happens the large subunit can be added on, this results in hydrolysis of GTP and now we have the initating complex. From there, the next tRNA can come in with the amino acid and sit in the A site, where the polypeptide chain can be built up, this is elongation.

19
Q

Steps in protein synthesis
Elongation of the polypeptide

where is polypeptide formed? hence where does it come out from?

where does incoming tRNA bind to? using what? (2)
hence what?

what happens to the polpeptide? how is this done?

after this is done, what happens? what does this require?

what are polysomes?

A

As the polypeptide is formed on the P site, it comes out form the top of the large ribosomal unit

Incoming aminoacyl tRNA attaches to the A site
on the ribosome using EF1 and GTP hydrolysis
- here the carboxyl group of the amino acid is joined to the tRNA and the amino group is free

The polypeptide from P site moves on top of the amino acid (on the amino group) on the A site
- this is done using the non protein enzyme peptidyltransferase
- this is a relatively small distance and so does
not require a lot of energy

• the ribosome now moves along the mRNA
- the polypeptide now moves to the P site, and
the consumed mRNA moves to the E site where it exits the ribosome
- all of this requires energy from GTP hydrolysis
and EF2

• usually, multiple ribosomes translate the message
- these are called polysomes: multiple groups of
ribosomes making multiple copies of one protein

20
Q

Steps in protein synthesis
Elongation of the polypeptide (different explanation)

where is the the polypeptide chain from the start? where does the next tRNA come to and what does this involve?

what happens next? what does this require?

what is the arrangment now with tRNA? where is the polypeptide?

what happens now? what does this require? (3)

what is the ;ast step? what do these do now?

what are polysomes?

A

In the diagram below the start is where the polypeptide chain already has 3 amino acids in the chain, this is sat in the P-site of the ribosome.

The next amino acid, that is determined by the codon on the mRNA, comes in as the aminoacyl tRNA to the A-site and this involves an elongation factor (EF 1), there is also hydrolysis of GTP as it is an energy dependent process.

What happens next is that the amino acid is not added onto the polypeptide, rather, the polypeptide is added onto the next amino acid. The enzyme that does this is called peptidyltransferase. So re-iterating, this enzyme transfers the growing peptide onto the new amino acid that has just been brought in.

Because of this, the tRNAs get distorted. The first tRNA is sat in the P site without being bound to the peptide chain and the second tRNA sits in the A site, now being bound to the peptide. However the peptide still sits in the P groove.

The next step is translocation (controlled by the enzyme translocase), where the ribosome moves one codon along the mRNA and this puts the new tRNA into the P site and the old tRNA into the E site. This step involves another elongation factor (EF 2) and another GTP hydrolysis.

The final step of elongation is the discharge of the used tRNA

This elongation process can be repeated to grow the polypeptide, with the discharged tRNAs picking up their appropriate amino acids again.

In most cells, each mRNA has quite a few ribosomes attached, so one message is synthesizing multiple parts of the proteins at the same time. These are called polysomes

21
Q

Steps in protein synthesis

Termination of polypeptide synthesis (3)

A
  • release factors on ribosome attach to stop codons
  • GTP hydrolysis takes place
  • the ribosome disassembles from the mRNA
22
Q

Steps in protein synthesis
Termination of polypeptide synthesis (different explanation)

what does the ribosome reach? (3 key examples)

what reocgnises this?
what takes place now?

A

Eventually the ribosome reaches the STOP codon, of which there are three (UAA, UGA, UAG).

When this happens, a release factor protein recognises this and using GTP energy hydrolyses the bond holding the tRNA to the peptide chain and it drops off.

23
Q

Energy Requirement for portein synthesis/translation

what is it for each process?

A

Charging tRNAs with amino acid —- 1 ATP per amino acid

Initiation of polypeptide synthesis —- nATP (as ribosome goes down to look for initiation codon) and GTP (when large subunit attaches)

Elongation of the polypeptide [addition of AA, one at a time]———- 2GTP per amino acid (one when AA binds to A site, one during translocation)

Termination of polypeptide synthesis [release of polypeptide]——- GTP (breaks bond between tRNA and polypeptide)

24
Q

There are 3 reading frames possible

what will determine this?
if shifts, what happens?

A
  • A change in the reading frame will result in a frame shift and completely different amino acids will be translated. This can occur if there are mutations.
  • Each reading frame translates to a completely different polypeptide sequence
  • The reading frame will be determined depending on the position of the starting AUG codon
25
Q

Limitations of Antibiotics Acting on Protein Synthesis (3)

if virus?
example of resistance? (2)
what may it inhibit?

A

No action on viruses, which use the host’s protein synthesis machinery

Resistance by various mechanisms, including alteration of target site or destruction of antibiotic

May inhibit protein synthesis in mammalian mitochondria, which have ribosomes like bacteria

26
Q

Difference between mammalian and bacterial ribosome

ribosome?
subunits?

A 
Prokaryotic
Ribosome - 70S
Large & small subunit - 50S & 30S
Large subunit components -  23S & 5S RNA (31 proteins)
Small subunit - RNA 16S (21 Proteins)
Eukaryotic
Ribosome - 80S
Large & small subunit - 60S & 40S
Large subunit components - 28S, 5.8S & 5S RNA
50 proteins
Small subunit - RNA 18S (33 Proteins)

MCD (22 decks)

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