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BCMB2901 > Part 2- > Flashcards

Part 2- Flashcards

16/03/2018 Starting from eukaryotic transcription

1
Q

What type of system is the trp operon system

A

Repressor

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2
Q

What happens if Trp is in the surroundings of a bacterial cell?

A

• If Trp in its surroundings, the bacterium doesn’t want to make Trp (most complicated amino acid to produce)  waste of energy
• When tryptothan is present, this co-repressor binds to this trp repressor protein which enables the binding of the Trp to the operator which stop RNA polymerase from binding to the operator  no production of the mRNA .
-Basally expressed- not completely turned off

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3
Q

Can bacteria make trp or can it only be collected in the environment?

A

o Bacteria take up tryptophan from the environment, but can also make it using enzymes coded by 5 genes within the trp operon

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4
Q

What happens if Trp is absent from the bacterial cell?

A

• In absence of tryptophan, trp repressor does nothing and doesn’t bind to the operator
o The repressor does not have bound tryptophan, and cannot attach to the operator site, allowing transcription of trp genes

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5
Q

Describe how Rifampicin works

A

• Rifampicin- semi-synthetic derivative that blocks the channel in RNA polymerase into which the RNA-DNA hybrid must pass, and so blocks elongation after 2-3 nucleotides have been added

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6
Q

What is an overview of the differences between eukaryotic transcription and prokaryotic transcription

A

• More complex regulation
o Three types of RNA polymerase depending on what the transcript is
o Complex promoter elements
• RNA processing
o Modifications to 5’ and 3’ ends of transcript
o Splicing out segments
• Spatial and time separation between transcription and translation
o Transcription occurs in nucleus
o Translation occurs in cytoplasm
o No time delay in prokaryotes between transcription and translation but there are in eukaryotes

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7
Q

What are the 3 types of RNA polymerase in the eukaryotic cell?

A

 RNA polymerase I
 RNA polymerase II (need loads of those)
 RNA polymerase III

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8
Q

What is the location of RNA polymerase I in the eukaryotic cell?

A

Nucleolus

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9
Q

What transcripts does RNA polymerase I produce?

A

18S (small ribosomal subunit), 5.8S and 28S rRNA (those two are part of large ribosomal subunit)
-Made as single transcript then cleaved to make different rRNA subunits

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10
Q

How many subunits does RNA polymerase I have?

A

14

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11
Q

What is the mass of RNA polymerase I?

A

514 kDa

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12
Q

What is the location of RNA polymerase II in the eukaryotic cell?

A

Nucleoplasm

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13
Q

What does RNA polymerase II produce?

A

mRNA precursors and snRNA

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14
Q

How many subunits does RNA polymerase II have?

A

12

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15
Q

What is the mass of RNA polymerase II?

A

588

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16
Q

What is the location of RNA polymerase III in the eukaryotic cell?

A

Nucleoplasm

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17
Q

What transcript does RNA polymerase III produce?

A

tRNA and 5S rRNA (large ribosomal subunit)

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18
Q

How many subunits does RNA polymerase III have?

A

17

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19
Q

What is the mass of RNA polymerase III?

A

693

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20
Q

On which DNA molecule are promoters located?

A

 Promoters are always on the same DNA molecule as the gene they regulate (cis-acting elements)

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21
Q

What are different types of promoters for RNA polymerase II and where are these usually located?

A
  • Can mix and match
  • Generally on 5’ side of start site
  • TATA box
  • CAAT box and GC box
  • Initiator element (Inr)
  • Downstream core promoter element
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22
Q

Describe the location of the TATA box and what happens when a mutation occurs

A

o Centred at -25 at the 5’ end of the transcription start site
o Mutation of a single base significantly impairs promoter activity

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23
Q

What does the subscript under a nucleotide letter indicate

A

o Number at bottom of nucleotide signifies the frequency % of the base at that position

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24
Q

Describe the location of the CAAT and GC box, as well as in which genes the GC box is usually found

A

o Located between -40 and -150
o Positions of these sequences vary greatly
o Can be found on either the template or coding strand
o GC box is usually found in genes that are always expressed so GC good as RNA polymerase recruited

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25
Q

Where is the initiator element located, when does it occur and why is it useful?

A

o Centred at +1
 Helpful because some promoter elements can be far away so when RNA polymerase needs help determining where the start site is it will look for the initiator element
o Can compensate for an absent or degenerate TATA box
o Defines the start site since other promoter elements are at variable distances from that site

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26
Q

Describe the location of the downstream core promoter element and when it is found

A

o Cantered at +30 downstream of the start site
o Commonly found in conjunction with the initiator element in genes that lack the TATA box
 Eukaryotic genes that are highly expressed would normally have TATA box with initiator element, or see initiator element followed by downstream element

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27
Q

What is the recognition difference between prokaryotic and eukaryotic promoters

A

 Unlike bacterial promoters, eukaryotic promoter elements are recognised by proteins (such as transcription factors) other than RNA polymerase itself

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28
Q

What are enhancer elements, where can they be and what do they do?

A

• Cis acting element that controls transcription
• These are DNA sequences stimulate transcription, but have no promoter activity of their own
• Can exert stimulatory action over a large distance (several thousand base pairs)
o Can recruit other proteins that can help stimulate transcription or proteins that then recruit RNA polymerase
o Don’t have to be next to promoter element due to large range
• Can be upstream, downstream, or in the middle of a transcribed gene
• Can be present on either DNA strand

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29
Q

What are transcription factors and are they present (besides the sigma factor) in prokaryotes?

A

• TFs are proteins that bind to promoter/enhancer elements and help recruit RNA polymerase
o TFII- the set of TFs that recruit RNA polymerase II
o In prokaryotic transcription, only RNA polymerase recognises promoter site- no transcription factors

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30
Q

How do transcription factors recruit RNA polymerase?

A

o Transcription factor binds to promoter box which induces a number of conformational changes within the protein
o Changes provide docking sites for additional transcription factors to come in
o Set of transcription factors can recruit RNA polymerase together
o TF complex unwinds DNA (so RNA polymerase can get access), recruits the RNA polymerase and phosphorylates the RNA polymerase, marking the transition from the initiation to elongation stage
o Other transcription factors are then ejected from machinery and the RNA pol can then transcribe the rest of the gene

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31
Q

How can RNA polymerase activity be promoted or repressed and what is this called?

A

• Basal transcription complex (bound to promoter region including RNA pol and transcription factors) initiates transcription at a low frequency
• Additional transcription factors bind to enhancer elements to increase transcription rate
• Transcription factors usually recruit other proteins (such as mediator/large protein- act as a bridge between transcription factors that are bound to enhancers and the RNA pol/transcription factors bound to the promoter because enhancer can be extremely far away from promoter) to build up large complexes that activate or repress transcription
o This entire set of proteins is known as the transcription machinery

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32
Q

How do transcription factors enable complexity of an organism?

A

• Transcription factors have tissue-specific expression, and can have different roles depending on what other proteins are present
o Allow for transcription of genes that are not normally expressed in other cells- specificity
• Combinatorial control of transcription forms the basis of functional complexity in eukaryotes
o Genes can be regulated in more sophisticated ways and can turn on or off depending on if certain transcription factors are present or not
o Function of individual transcription factors change depending on if other transcription factors are present

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33
Q

What is oestrogen?

A

• Oestrogens- cholesterol derived steroid hormone that regulate ovarian cycle
o Hydrophobic molecule

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34
Q

How does oestrogen promote transcription of gene?

A
  • They diffuse into cell (because hydrophobic) and bind to oestrogen receptor (a type of nuclear hormone receptor) soluble in the cytoplasm or nucleoplasm
  • Ligand-binding induces conformational change in particular domain which will help recruit transcription factors (coactivators)
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35
Q

Describe the structure of a nuclear hormone receptor

A

• The nuclear hormone receptor has ligand-binding and DNA-binding domains and binds to them
o Zn finger domain recognises specific sequences of DNA
o Ligand binding domain binds ligand in hydrophobic pocket that causes structural change in domain that can recruit proteins to promote transcription

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36
Q

What is a zinc finger domain?

A

o A zinc finger is a small, functional, independently folded domain that coordinates one or more zinc ions to stabilize its structure through cysteine and/or histidine residues.

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37
Q

How do receptor ligand complexes work with ligands?

A

o Receptor binds as a dimer and their DNA binding domain binds to specific sequence that it recognises- ligand binding does not affect DNA binding (so when ligand not bound can still bind to DNA)
o Ligand binds to ligand binding domain- induces structural change- recruits proteins called coactivators or corepressors

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38
Q

What is a coactivator?

A

protein that can help recruit RNA polymerase or other transcription factors and enhance transcription

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39
Q

What is a corepressor?

A

molecules that stop the recruitment of transcription factor or RNA polymerase and inhibit transcription

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40
Q

What is an agonist?

A

molecule that can bind to receptor and trigger signalling

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41
Q

What is an antagonist?

A

bind to receptor but stop signalling from happening

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42
Q

What do agonists of the androgen receptor do?

A

• Agonists of the androgen receptor stimulate expression of genes that enhance development of lean muscle mass

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43
Q

What do antagonists of oestrogen receptors do?

A

• Antagonists of oestrogen receptors e.g. tamoxifen and raloxifene can stop oestrogen-mediated cell growth and are used to treat some breast cancers

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44
Q

How is rRNA processed and made?

A

• Transcribed by RNA Pol I as a single 45S precursor- processing occurs in nucleolus of cell
1. Nucleotides are modified
 Modified at base group or ribose group by small ribonucleoproteins
2. Pre-rRNA is assembled with ribosomal proteins
3. Pre-rRNA is cleaved into 18S (small part of subunit), 28S and 5.8S rRNA (large ribosomal subunits)
• 5S rRNA is transcribed separately by RNA Pol III

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45
Q

How is tRNA processed and made?

A

• Transcribed by RNA pol III
1. Nucleotides from 5’ and 3’ ends are cleaved
 5’ leader and 3’ trailer are cut off
2. Nucleotides CCA are added to 3’ end
3. Nucleotides are modified on base and ribose groups
4. Intron is removed and products are ligated

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46
Q

In bacteria does transcription and translation occur simultaneously in the same space? Is it the same for eukaryotes?

A
  • In bacteria, transcription and translation occur simultaneously in the same space
  • In eukaryotes, pre-mRNA transcripts are extensively processed into the cytoplasm before translation
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47
Q

Does mRNA processing occur in prokaryotes?

A

• mRNA processing is EXCLUSIVE to eukaryotes

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48
Q

What are the 3 major mRNA processing events?

A
  1. Capping at 5’ end
  2. Polyadenylation
  3. Splicing
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49
Q

When does 5’ capping mRNA occur?

A

 Occurs when transcript is about 25 nucleotides long

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50
Q

What is the end result of 5’ capping mRNA and how is this achieved?

A

 Capping adds a methylated guanine to the transcript via a 5’-5’ linkage
 Done by 3 enzymatic reactions
• Pre-mRNA transcript has 3 phosphates at 5’ end.
• One of those phosphates is removed by phosphatase
• Left with diphosphate group, which attacks alpha phosphate group of GTP
• GTP added by guanylyl transferase which transfers a guanosine mono phosphate group, releasing pyrophosphate, to form 5’ to 5’ phosphate linkage
• Methyl transferase methylates guanine

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51
Q

What is the purpose of 5’ capping of mRNA?

A

 Capping protects the 5’ end of mRNA from phosphatases and nucleases and enhances mRNA translation

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52
Q

When does polyadenylation occur in mRNA?

A

After transcription has ended

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53
Q

Is the poly (A) tail of mRNA encoded by DNA?

A

NO

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54
Q

How does polyadenylation of mRNA happen?

A

 Pre-mRNA is cleaved and around 250 adenylate residues are added using ATP as the substrate
• Cleavage signal is AAUAAA
• Complex that contains endonuclease will go forth and look for this signal
• Once it finds it, it cleaves pre-mRNA somewhere after that cleavage signal
• After cleavage occurs, poly(A) polymerase comes in and adds on a series of A nucleotides at the 3’ end of this transcript: uses ATP as the A donor

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55
Q

What is the purpose of a polyA tail in mRNA?

A

 Poly(A) tail increases mRNA stability and enhances rate of translation

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56
Q

How does a polyA tail in mRNA increase stability of mRNA?

A

o 3’ poly-A tail binds specific proteins which then interacts with other proteins that stop deadenylase enzymes (that normally degrade poly-A tail) and stop cleavage enzymes from cutting off mRNA

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57
Q

What happens if mRNA has no poly(A) tail?

A

 Deadenylation (no 3’ poly(A) tail) is associated with mRNA decay

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58
Q

Which eukaryote mRNA is not polyeadenylated?

A

Histone mRNAs

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59
Q

How are histone mRNAs processed?

A

 Histone mRNAs have a stem-loop structure followed by a purine-rich sequence to direct cleavage after where the stem loop occurs
• Only form of processing that occurs to histone mRNAs

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60
Q

What percentage of human genes have both introns and exons?

A

 >90% of human genes have exons (coding regions) and introns (non-coding regions)

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61
Q

How many nucleotides long can introns be?

A

 Introns can be 50-10,000 nucleotides long

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62
Q

What is splicing in mRNA?

A

 Splicing removes the introns and links the exons to form the mature mRNA (only happens in eukaryotes)

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63
Q

Describe the composition of an intron and what their purposes are

A

 Consensus sequences at the ends of introns identify splice sites
• 5’ GU splice site
• 3’ AG splice site
• 3’ Pyrimidine trap
o 10 U/C in the region followed by any nucleotide followed by C
o Part of 3’ splice site
 Branch site- located 20-50 nucleotides upstream of a 3’ splice site
• In intron with A somewhere in the middle
 These sequences will define where and how splicing occurs

64
Q

Are mutations in in intron splice sites/branch sites significant?

A

 Mutations in splice sites or branch sites lead to aberrant splicing
• Mutations can shift entire reading frame -stop codon can occur prematurely

65
Q

What is a spliceosome?

A

large splicing complex (around 4.8 MDa) consisting of small nuclear RNAs (snRNAs) combined with more than 300 proteins and pre-mRNA

66
Q

What is a snrnp?

A

o Combination of snRNAs with proteins is called small ribonucleoproteins (or snrnp)
 Given names such as U1

67
Q

How does intron splicing occur?

A

o U1 and U2 snrnp come into pre-mRNA
o U1 binds to 5’ splice site and U2 binds to branch sites
o U4-U5-U6 complex comes in and joins U1, U2 and pre-mRNA which forms the complete spliceosome
o U6 and U2 interact with each other which ejects U1 and U4 out of spliceosome complex
o First transesterification occurs:
 A in branch sites attacks 5’ splice site which releases first exon so that it is no longer connected to the rest of the transcript (but still held in place- not ejected out of spliceosome)
 Intron becomes a lariat intermidate
o Second transesterification process occurs:
 5’ splice site free -OH group attacks the 3’ splice site which forms linkage between exon 1 and exon 2
 Rest of intron (still as a lariat intermediate) gets released with rest of the snrnps

68
Q

Why are the snRNAs in the spliceosome important?

A

o The snRNAs present within spliceosomes are the ones responsible for aligning splice site and catalysing a lot of the reactions

69
Q

What do helicases do in the spliceosome?

A

o Helicases powered by ATP that unwind RNA duplex which then releases your snrnps and intron lariate leaving the two exons ligated together

70
Q

Mutations affecting splicing cause what percentage of all genetic disease?

A

15%

71
Q

Where can mutations affecting splicing occur?

A

• Mutations can occur in the pre-mRNA (e.g. thalassemia) or in the splicing factors

72
Q

Describe how thalessimia occurs

A

 Stop codon normally taken out and doesn’t cause a problem

 But if the mutation occurs then stop codon doesn’t taken out, leading in a a truncated protein

73
Q

How can the poly(A) tail purify mRNA from total RNA? Describe the method

A

• mRNA can be purified from total RNA using Oligo-dTs that bind to the poly(A) tail
1. Mixture is made:
 Cultured cells
 Animal/plant tissue
 Cells immobilised on cell-specific Dynabeads
 Serum/plasma
 Total RNA
2. Add Dynabeads Oligo to crude lysate containing polyadenylated RNA
3. Hybridize and wash
 3’poly(A) tail binds to oligdTs
4. Can elute everything that doesn’t contain this polyAtail (leaving only mRNA)
5. Separate out the strands to collect just mRNA or can process mRNA whilst its bound to beads: can do reverse transcription using oligodT as a primer

74
Q

What is alternative splicing and what is its implication?

A
  • Can have exons that have a choice as to whether they’re incorporated in the final mRNA product or not- called alternative splicing
  • One gene but many mRNAs= many proteins
75
Q

What percentage of human protein-coding genes are alternatively spliced?

A

• >70% of human protein-coding genes are alternatively spliced

76
Q

How can you tell how many different mRNAs can result from one pre-mRNA?

A

• 2n different mRNAs from one pre-mRNA with n alternative splice sites

77
Q

What is an example of alternative splicing?

A
  1. Alternative splicing gives rise to membrane-bound and secreted variants of the same antibody
    - Membrane-anchoring unit encoded by a seperate exon: results in membrane bound antibody
    - Alternative splicing of RNA excludes membrane-anchoring domain: antibody is secreted into extracellular medium
78
Q

Are transcription and mRNA processing in eukaryotes coupled?

A

Yes

79
Q

How are transcription and mRNA processing in eukaryotes coupled?

A
  • RNA Pol II, once c terminal domain is phosphorylated, recruits capping enzymes, splicing machinery components, and polyadenylation enzymes
  • Once in about 25 nucleotides are transcribed, capping enzyme is recruited and puts that cap on
  • Once the first intron is made, splicing enzymes are recruited and each intron is spliced as it is produced
  • Once transcription is finished, you get polyadenylation enzymes that makes the polyA tail
80
Q

What happens to mature mRNA after processing?

A

• After processing, mature mRNA is transported from the nucleus to the cytosol through the nuclear pore complex where it is translated

81
Q

What is a nuclear pore complex?

A

• Nuclear pore complex- pores in nuclear membrane that allow water soluble molecules to pass through

82
Q

In average eukaryotic cell, how many nuclear pore complexes are there and how many transports per second?

A

• In average Eukaryotic cell- up to 2000 nuclear pore complexes and get 1000 transports per second

83
Q

Describe the differences in structure between nucleic acids vs amino acids, and why this is so

A

• Nucleic acids: 4 common bases
o Fairly similar structure
o Made up of carbon-nitrogen ring system
o Only responsible for encoding information
• Amino acids;
o Diversity of structures
o Reason: molecules that perform functions to sustain life
• Because of difference in function, underpins structural diversity

84
Q

Is the genetic code universal?

A

• Nearly universal
o Nearly same set of genetic code in all organisms
o Why you can put human genes in bacterial plasmids

85
Q

What is a codon and why do we need it to be a codon?

A

• A set of 3 nucleotides, called a codon, encodes an amino acid
o There are 4 types of nucleotides, so need at least 3 to code for 20 amino acids

86
Q

Are codons overlapping or non-overlapping

A

non-overlapping

87
Q

Does the genetic code have directionality?

A

Yes- 5’ to 3’

88
Q

Is the genetic code degenerate? Why?

A

• Is degenerate
o 3 nucleotides= 64 distinct codons but only 20 amino acids, so most amino acids are encoded by >1 codon.
o Degeneracy minimises the bad effects of genetic mutations
 Having mutations at DNA level are less likely to be transferred as a mutation at the protein level

89
Q

What is a synonymous mutation?

A

 Synonymous mutation- DNA mutation encodes for the same amino acid at the protein level

90
Q

What is the risk of translation error and what is the limit that this risk has to be managed at?

A

• Translation is complex-potential for error at each step
• Need to balance translation accuracy with speed (40 amino acids/sec in E.Coli)
• How accurate must translation be?
o In E.Coli, average protein has 300 amino acids
o Some have >1000 amino acids
o So translation error frequency must not exceed 1 per 10,000 amino acids
 This is about the error rate in E.Coli

91
Q

Why are transfer RNAs adaptor molecules?

A
  • Adaptor molecules because they recognise the codon

* Transfer RNA (tRNA) binds to a specific codon and brings an amino acid with it

92
Q

What is the basic structure of tRNA’s and what is the result of this?

A

• At least one tRNA for each amino acid (attached to 3’CCA) with anticodon in a central loop
• Single-stranded, with 73-93 ribonucleotides, many modified and base-paired to form an L-shaped structure, including loops and helices (because of base pairing between nucleotides of tRNA)
o Different types of tRNA have similar structural features as they have to interact with same machinery (mRNA and ribosomes)
o Loops and helices are what differentiate each type of tRNA apart from each other

93
Q

Describe codon and anticodon simple base pairing and what it falsely suggests

A

o Simple base pairing (simplest case as to what could happen)
 Codon and anticodon line up in antiparallel manner (1st base pair of codon base pairs to 3rd base pair of anticodon and so on)
o This suggests each anticodon binds to only one codon, which is not always the case, so don’t get simple base pairing in all nucleotides of tRNA

94
Q

Why is simple codon-anticodon base pairing unrealistic in all nucleotides of the codon? What happens instead and why?

A

o Some tRNAs recognise more than one codon
 Recognition of the 3rd base in the codon is less discriminating than the first two
 There is a steric freedom in the 3rd base of the codon pairing between the mRNA codon and the tRNA anticodon
• Not simple Watson-Crick pairing
 The 2nd and 3rd base in anticodon pair with the codon in the standard way (Watson-Crick manner) , while the 1st base of the anticodon determines if tRNA reads 1,2 or 3 types of codons
• Because codons that code for same amino acid usually have a change at their 3rd base

95
Q

list the first bases of anticodons, and what their potential third bases are:

A 
First base of anticodon	(first row)
Third base of codon (second row) 
C	                G
A	                U
U	                A or G
G	                U or C
I (inosine)	U, C, or A
96
Q

What does binding of an amino acid to tRNA do?

A
  • Binding of an amino acid to a particular tRNA establishes the genetic code
  • Binding of amino acid to tRNA activates the amino acid
97
Q

Why is activation of the amino acid necessary and how does it happen?

A

o Activation needed because formation of peptide bond between free amino acids is thermodynamically unfavourable
 Activation process is when your amino acid is bound at its carboxyl group to an -OH belonging to the adenine of the CCA arm of tRNA
o Activation  amino acid ester
o Activation done by specific aminoacyl-tRNA synthetase enzymes using 2 ATP
 Amino acid+ ATP+ tRNA+ H2O  aminoacyl-tRNA+ AMP+ 2Pi

98
Q

Are aminoacyl-tRNA synthetases the same for every amino acid and tRNA? How/how not?

A

• Aminoacyl-tRNA synthetase must put the correct amino acid onto the tRNA
o Must be able to recognise the specific amino acid for that specific tRNA
o Each enzyme is highly specific for a given amino acid
 Many different types of aminoacyl-tRNA synthetase enzyme
o Enzyme uses the specific properties of its amino acid substrate

99
Q

Describe how threonyl-tRNA synthetase ensure specificity/how it works

A

 Threonyl-tRNA synthetase:
• Threonine is its substrate which can bind to the active site via interactions with zinc ion and asp group that this enzyme has
• These reactions give specificity to this process
• Zinc/Asp mechanism can differentiate Threonine and Valine (cannot bind to valine) but not Threonine and Serine (because of same binding group)
o E.g. Presence of editing site which accepts serine attached to threonyl-tRNA and breaks the bond between serine and the tRNA
 CCA arm is flexible: can swing between editing site and activation site
 Serine fits into editing site and is cleaved from the threonyl-tRNA
 Arm moves back to activation site and correct residue gets chance to get right amino acid to get put on
 Extra methyl group makes threonine too big to fit into editing site

100
Q

Why does aminoacyl-tRNA synthetase have a proofreading function?

A

• Aminoacyl-tRNA synthetase has proofreading function to prevent these discrimination mistakes from occurring

101
Q

Explain the double-sieve mechanism of aminoacyl-tRNA synthetase

A

 Activation site usually excludes amino acids that are larger than correct amino acid
 Editing site usually excludes amino acids that are smaller than correct amino acid
 Double-sieve function ensures high fidelity of the process

102
Q

How does aminoacyl-tRNA synthetase choose the correct tRNA partner?

A
  • Aminoacyl-tRNA synthetase must choose the correct tRNA partner
  • The correct tRNA is recognised via its anticodon, its unusual/modified bases, and its structure (loops and helices)
103
Q

What are ribosomes composed of?

A

• Composed of RNA and proteins that coordinate the interplay of mRNA, tRNA and proteins for proteins synthesis

104
Q

What makes the catalytic site of ribosomes?

A

• Key catalytic sites are mainly composed of RNA, with minor contributions from proteins

105
Q

What subunits are bacterial ribosomes composed of?

A

• In bacteria, the 70S ribosome is composed of large (50S) and small (30S) subunits

106
Q

What do ribosomes bind?

A

• Ribosomes bind tRNA and mRNA

107
Q

How many tRNA binding sites do ribosomes have and what are they called? How are mRNA and tRNA bound within them?

A
  • Ribosomes have 3 tRNA-binding sites that span the 50S and 30S subunits: the Aminoacyl, Peptidyl (where protein synthesis is elongated) and Exit sites
  • mRNA is bound within the 30 S subunit
  • tRNA in the A and P sites are bound to mRNA via anticodon-codon pairing
108
Q

Are prokaryotic transcription and translation closely coupled in space and time? How so and why is this possible?

A
  1. Prokaryotic transcription and translation are closely coupled in space and time
  2. The 5’ end of mRNA interacts with ribosomes way before transcription of the 3’ end is finished
     Possible because both transcription and translation proceed in the 5’ to 3’ direction
  3. Multiple ribosomes translating and mRNA strand
109
Q

Where is the first translated codon commonly located in bacteria?

A

 Translation does not begin at the start of the mRNA

 The first translated codon is nearly always more than 25 nucleotides away from the 5’ end

110
Q

Do mRNAs in bacteria always only contain one gene or many?

A

 In bacteria, many mRNA are polycistronic

1. Each gene has its own translation start and stop signals on mRNA

111
Q

What are the translation start signals on mRNA?

A
  • Initiator codon

- Shine-Dalargano sequences

112
Q

Describe what Shine-Dalgarno sequences are

A

purine-rich sequence centred about 10 nucleotides upstream of the initiator codon

113
Q

Why are Shine-Dalagarno useful?

A

o Binds to 16S rRNA in the ribosomal small subunit
o Aligns mRNA so that tRNA so that first tRNA can come in and bind at the right spot- so binding is very important between Shine-Dalagarno sequence and 16S rRNA

114
Q

What is the initiator codon in translation?

A

AUG (methionine)

-Binds to the anticodon of the first tRNA

115
Q

Describe the initiator tRNA and its corresponding amino acid in bacterial translation

A

o Bacterial protein synthesis starts with a modified version of methionine, N-formylmethionine (fMet)
 The initiator tRNA recognises the AUG initiator codon and brings in fMet
 The initiator tRNA is distinct from the tRNA that inserts methionine in internal positions

116
Q

Are all methionines modified?

A

 Only the methionine associated with the initiator tRNA is modified

117
Q

Is fMet always present in the final polypeptide product?

A

 fMet is removed from about half of all proteins once the new polypeptide chain is about 10 amino acids long

118
Q

How is fMet for bacterial translation produced?

A

• Methionine is linked to the initiator tRNA and the normal Met tRNA by the same aminoacyl-tRNA synthetase
o Same enzyme puts unmodified methionine on both initiator tRNA and normal tRNA
o Puts methionine via its carboxyl group onto that 3’-OH group that is in CCA arm of tRNA
 Esterification process activates the amino acid
• Modified when attached to tRNA that carries it
• A specific enzyme, transformylase, formylates the methionine attached to the initiator tRNA (modifies the methionine group by adding a formyl group to the end of the methionine)
o This enzyme cannot formylate free methionine or methionine attached to the normal Met tRNA (only to the initiator tRNA)
 Ensures that only methionine that is attached to initiator tRNA that gets modified

119
Q

Describe how initiation of translation occurs in bacterial cells

A
  1. Initiation factors (IFs-set of proteins that help with initiation of translation) bind to small 30S ribosome subunit (2 of them to be exact) and keeps it apart from the large 50S subunit
  2. mRNA containing Shine-Dalgarno sequence will come in and bind to small ribosomal subunit via 16S rRNA
    o 16S rRNA within the ribosome and the Shine-Dalgarno sequence within mRNA positions your mRNA in the right place for the right place
  3. Binding of initiator tRNA (with fMet)
    o Comes in and binds to start codon of mRNA
    o Chaperoned in place by initiation factor called IF2
     IF2 binds no other tRNA except initiator tRNA
     Makes sure the other methionine tRNA does not accidently get slotted in that position
  4. IFs, initiator tRNA, mRNA and 30S subunit form the 30S initiation complex
    o mRNA binds to rRNA in 30S
    o tRNA binds to AUG codon in mRNA and to the P site in 30S
  5. Once initiation complex is formed, there are structural changes that occur within this complex which eject some of the initiation factors (like IF1 and IF3) but the remaining initiation factor (IF2) will recruit large ribosomal subunit (50S)
  6. 50S subunit binds to 30S initiation complex to form 70S initiation complex
    o Rate-limiting step
    o Signals the progression from initiation to elongation
120
Q

Describe elongation in bacterial translation

A

 Starting point: ribosome P site has initiator tRNA, A and E sites are empty
 Binding of first tRNA to start codon establishes reading frame
 Next tRNA is delivered to A site by elongation factor Tu (EF-Tu)
1. EF-Tu protects ester bond in activated amino acid from hydrolysis
2. EF-Tu checks accuracy of anticodon-codon binding before it leaves
3. EF-Tu binds to all tRNA except initiator tRNA
o Makes sure that normal tRNA carrying unmodified methionine when AUG codon appears internally
 At this point, P and A sites are occupied
 fMet from initiator tRNA is transferred to amino group of amino acid in A site
 Formation of peptide bond (now spontaneously advantageous as amino acids are activated) is catalysed by rRNA in the large 50S ribosomal subunit
1. Catalysis by proximity and orientation
2. Uses proximity and orientation to take advantage of inherent reactivity of amine group (on amino acid in A site) with an ester (on initiator tRNA in P site)
3. Amino acid is transferred from the tRNA in the P site to the amino acid on the A site
o Amino group on the amino acid on the A site will launch a nucleophilic attack against that ester linkage that binds your amino acid and your tRNA in the P site
o Transition state is formed
o Transition state collapses and you get a peptide bond between 2 amino acids
 N terminus of your incoming amino acid will be linked to the C terminus of your growing polypeptide chain

121
Q

Describe translocation in bacterial translation

A
  1. At this point, peptide chain is attached to tRNA on A site
  2. Ribosomal subunits rotate with respect to each other
  3. That rotation means that the peptide chain that is originally on the A site gets transferred to the P site but only in the large subunit- peptide chain is still bound to the tRNA that is on the A site in the small subunit
    o Ribosomal subunits rotate such that this peptide-tRNA is in the A site of the small ribosome subunit but in the P site of the large ribosome subunit
  4. Process stalls for a bit as the process can no longer continue until mRNA can be moved by 3 nucleotides so that the new codon is in the A site ready to accept new incoming tRNA
  5. The elongation factor G (EF-G) helps move the tRNA and mRNA by 3 nucleotides in the small subunit, a process called translocation
    o The peptide-tRNA (contains growing peptide chain) moves from the A site into the P site (in the small subunit-now they’re aligned again)
    o The empty tRNA (transferred its amino acid to the growing chain) moves from the P site to the E site (in the small ribosomal subunit-aligned again with big rRNA)
    o The next codon is positioned into the A site- now ready to accept new incoming tRNA
  6. The tRNA in the E site is released by the ribosome
    o tRNA with growing polypeptide chain in the P site, A site and E site now empty
  7. Proteins are made in the N to C terminal direction (N terminus of new amino acid is put on C terminus of existing polypeptide chain)
    o Opposite to how we make proteins in the lab- we go from last to first
122
Q

Describe the termination process of protein translation in bacteria

A
  1. RFs bind to stop codon and P site on large subunit
    o RFs bind to stop codon (which are on A sites of ribosome) and bridges the gap between your A site and your P site, which is where your growing polypeptide chain is located
  2. RF has glutamate residue in it that is modified, promotes breaking of ester linkage between tRNA and polypeptide chain through a water molecule attack at the ester linkage
  3. Polypeptide leaves the ribosome
  4. Complex of tRNA, mRNA and ribosome dissociates
123
Q

What are the stop codons and what happens when these are reached?

A

 Stop codons: UAA, UGA, UAG
 No tRNAs with anticodons complementary to Stop codons
 Stop codons are recognised by release factors (RFs-proteins that help with termination process)

124
Q

What are the differences in ribosomes between eukaryotic and prokaryotic translation?

A
  1. Ribosomes- eukaryotic ribosomes are larger
     80S (eukaryotes) vs 70S (bacteria)
     Each subunit in eukaryotic ribosomes have more proteins and bigger mRNA components
125
Q

What are the differences in initiator tRNA between eukaryotic and prokaryotic translation?

A

 In eukaryotes, starting amino acid is methionine rather than N-formylmethionine
 Like in bacteria, a special tRNA participates in initiation

126
Q

What are the differences in initiation between eukaryotic and prokaryotic translation and why?

A

 In eukaryotes, translation is decoupled from transcription
 No Shine-Dalagarno sequence in eukaryotic mRNA to signal initiation of translation
• Bacteria need these due to their polycistronic nature so it can identify the start site-Shine-Dalagarno sequence indicates that the next AUG seen is the starting point for that gene
• Eukaryotic- most encode for only one protein, so not need to tell if internal AUG is start site for a particular protein
 First AUG of eukaryotic mRNA is usually selected at start site for translation since the mRNA encodes only one protein product
• The 40S ribosome, initiator tRNA (with unmodified methionine) and eukaryotic initiation factors form a complex
• Complex binds to 5’ cap of mRNA and searches for AUG codon with help from helicases powered by ATP
• Initiator tRNA anticodon binds to AUG on mRNA
• Translation proceeds

127
Q

What are the differences between structure of mRNA in prokaryotic and eukaryotic translation?

A
  1. Structure of mRNA
     Eukaryotic mRNA is circular, presumably to prevent translation of mRNA without poly(A) tails (to make defective mRNA without a poly(A) tail isn’t being translated)
     Circular because EIFs (eukaryotic initiation factors) bridge your 5’ cap and your 3’ Poly A tail together to form that circular structure
128
Q

What are the differences between prokaryotic and eukaryotic translation in terms of elongation and termination?

A

 Bacterial elongation factors and release factors have eukaryotic counterparts
• E.g. Eukaryotic EF2= bacterial EF-G

129
Q

What are the differences between prokaryotic and eukaryotic translation in terms of organisation?

A

 In eukaryotes, components of the translational machinery are organised into large complexes associated with the cytoskeleton
 Organisation believed to facilitate efficiency of protein synthesis and compartmentalise the translation process within your cell

130
Q

What do streptomycin and other aminoglycosides do?

A

Prevents binding of fMet-tRNA to ribosomes (specific to bacteria)
Inhibit initiation and cause the misreading of mRNA (bacteria)

131
Q

What does tetracycline do?

A

Binds to the 30S subunit and inhibits the binding of aminoacyl-tRNAs (bacteria)

132
Q

What does chloramphenicol do?

A

Inhibits the peptidyl transferase activity of the 50S ribosomal subunit (bacteria)

133
Q

What does cycloheximide do?

A

Inhibits translocation (eukaryotes)

134
Q

What does erythromycin do?

A

Binds to the 50S subunit and inhibits translocation (bacteria)

135
Q

What does puromycin do?

A

Causes premature chain termination by acting as an analog of aminoacyl-tRNA (bacteria and eukaryotes)

136
Q

How does toxin from C.diphtheria cause diphtheria?

A

 Toxin adds ADP-ribose to diphthamide, an essential amino acid in EF2. This blocks EF2’s ability to carry out translocation of the growing polypeptide chain and stops translation
 Extremely potent
 Process:
• Diphtheria toxin’s receptor-binding domain (B) binds host membrane
• Membrane-bound toxin (A+B) enters by endocytosis
• Catalytic subunit A is cleaved but held to the B subunit by disulphide bonds. Endosome vesicle acidifies: the disulfide bonds are reduced
• The transmembrane domain facilitates passage of the catalytic A peptide through the vesicle membrane
• The catalytic A domain ADP-ribosylates elongation factor 2 (EF2). This halts protein synthesis and kills the cell

137
Q

How does ricin from castor beans stop protein translation?

A

 Toxin removes adenine from an adenosine on 28S rRNA. This inactivates ribosomes and prevents binding of elongation factors

138
Q

How was IRES discovered and how was it discovered to work?

A

o First IRES site found in poliovirus
o Virus secrets a particular protease that chops off one of eukaryotic initiation factors, which means it can’t help in initiation of normal eukaryotic mRNA
o Virus mRNA (which has IRES site) only needs a fragment of that initiation factor to start translation process
o To combat this, some eukaryotic mRNAs also contain IRES sites which allows translation even when viral infection chops of the essential initiation factors involved in cap process (backup)

139
Q

What is the IRES and what does it do?

A

• These mRNAs have an internal ribosome entry site (IRES) on 5’ side of the start codon
o Not part of start codon but upstream of it
o Part of 5’ untranslated region of mRNA
• The IRES interacts with the 40S ribosome subunit or the initiation factor elF4F to start translation

140
Q

Describe how IRES can be used experimentally methodically

A

• Experimental application of IRES-
o Allows co-expression of several genes under the control of the same promoter
o You can introduce IRES downstream the gene of interest and preceding a reporter gene
o IRES allows us to add in gene sequences that will be under the control of endogenous promotors; will be transcribed every time the gene of interest is transcribed
o Allows for the independent translation of genes under the same promoter
 This is practical as fusing proteins together, which would normally happen, can change the function of proteins of interest/the report, so their independent production is an advantage

141
Q

Describe how IRES can be used experimentally in terms of uses

A

o Used for:
 Finding where the gene of interest is being expressed (due to the independent translation of the reporter gene
 Can monitor upregulation/downregulation analysis

142
Q

What are 3 types of mRNA degradation?

A
  • Nonsense mediated mRNA decay
  • Non stop mRNA decay
  • No go mRNA decay
143
Q

Describe how nonsense mediated mRNA decay occurs

A

 A process that detects and destroys transcripts with a premature stop codon
• During pre-mRNA splicing, the site of each excised intron (the boundary between exons) is marked by an exon junction complex (EJC)
• Ribosome removes EJCs as it moves along and translates the mRNA
• If a premature stop codon is present, the ribosome is released early and some EJCs remain on mRNA
• EJCs recruit proteins such as B-capping enzymes that cleave the 5’ cap
o mRNA is degraded by RNases

144
Q

Describe how non-stop mRNA decay occurs

A

 Without a stop codon, translation proceeds through the 3’ poly(A) tail
• AAA encodes lysine
 Stalled ribosome binds a protein that triggers ribosome dissociation and mRNA degradation by 3’ 5’ RNase, and also recruits the protease
 Defective polypeptide is degraded by a protease that recognises the C-terminal poly(lysine) tag

145
Q

How does no go mRNA decay occur

A

 Stalled ribosome recruits factors that promote ribosome dissociation, mRNA cleavage and mRNA degradation

146
Q

Why do ribosomes stall during translation?

A

 Ribosome stalls before the stop codon is reached: could be due to:
• Rare codons
o When a codon is rare, the tRNA for it is rare too, and hence the ribosome has to stall to wait for the rare tRNA to show up
• Secondary structure
o Such as stem loop structures

147
Q

What is RNA interference?

A

process of mRNA degradation induced by double-stranded RNA (dsRNA)

148
Q

Where does dsRNA come from?

A
  • Exogenous(e.g. viruses; experimentally introduced)

- Endogenous(generated from larger transcripts made by RNA polymerase II/III)

149
Q

What happens to exogenous dsRNA in the cell?

A

o Exogenous (e.g. viruses; experimentally introduced)- known as siRNAs
 Exogenous dsRNA is cleaved by an Rnase called Dicer into 21-23 nucleotide long fragments
 Single stranded cleavage products called small interfering RNA (siRNA) bind Argonaute proteins to form RNA induced silencing complex (RISC)
• In the cytoplasm the sense strand of siRNA Is degraded and antisense strand forms a complex with RISC
 RISC binds to complementary target mRNA, which stops translation and/or causes mRNA degradation by recruiting enzymes that chop up the RNA

150
Q

What happens to endogenous dsRNA in the cell?

A

o Endogenous (generated from larger transcripts made by RNA polymerase II/III)- known as miRNAs
 MicroRNAs (miRNAs) are generated from larger transcripts made by RNA Pol II or RNA Pol III
 Endogenous dsRNA is cleaved by an Rnase called Dicer into 21-23 nucleotide long fragments
 Single stranded cleavage products called miRNAs bind Argonaute proteins to form RNA induced silencing complex (RISC)
 RISC binds to complementary target mRNA, which stops translation and/or causes mRNA degradation by recruiting enzymes that chop up the RNA

151
Q

What are the advantages/disadvantages of having iron in the cell?

A

• Context: Cellular iron (Fe) levels must be tightly controlled
o Fe is needed for synthesis of many proteins e.g. haemoglobin, and is a key part of making ATP
o But excess Fe can damage proteins, lipids, nucleic acids via free-radical reactions

152
Q

What does transferrin do?

A

Carries Fe in blood

153
Q

What do transferrin receptors do?

A

binds Fe+ transferrin and enables entry into cells

154
Q

What does ferritin do?

A

stores Fe in liver and kidney

155
Q

What is the relation between transferrin receptor production and ferritin production?

A

o Ferritin and transferrin receptor levels are reciprocally related
o Low Fe in cells means that transferrin receptor synthesis goes up but ferritin goes down
o High Fe in cells means that transferrin receptor synthesis goes down but ferritin goes up
o Rate of transcription does not change- regulation takes place during translation

156
Q

How is ferritin synthesised and regulated?

A

o Ferritin mRNA has a stem-loop called the iron response element (IRE) in the 5’ untranslated region which binds to the IRE protein
o When iron concentration is low, IRE binding protein binds to the IRE in ferritin mRNA and blocks the initiation of translation
o When iron concentration is high, IRE binding protein binds to iron and hence cannot bind to the ferritin IRE mRNA loop
 Ferritin mRNA is translated and ferritin stores excess iron

157
Q

How is transferrin synthesised or regulated?

A

o Transferrin receptor mRNA also has several IRE regions in the 3’ untranslated region that bind to the IRE binding protein
o Positioning of these IRES means that mRNA can still be translated even when the IRE binding protein is bound
o When iron levels are low, IRE binding protein binds to transferrin receptor mRNA
 Translation proceeds as normal, and the mRNA is protected from degradative enzymes due to the IRE binding protein
o When iron levels are high, IRE binding protein binds to iron and detaches from the transferrin receptor mRNA
 Therefore, mRNA is rapidly degraded by degradative enzymes and is not translated

BCMB2901 (4 decks)

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