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1
Q

Where does the following all occur in prokaryotes:

Replication, transcription and translation

A

It all occurs in the cytoplasm

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2
Q

What reasons are there for why the intermediate mRNA is required?

A
  1. Better regulation of the protein production process.
  2. Fidelity of DNA replication is very important. Having an intermediate step prevents interference and potential additional mistakes.
  3. Proteins can still be made when DNA is being replicated.
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3
Q

Is RNA linear?

A

RNA is a linear polymer like DNA

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4
Q

What links the residues in RNA?

A

phosphodiester bonds

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5
Q

What sugar does RNA contain?

A

ribose

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6
Q

Is RNA single or double stranded?

A

single stranded

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7
Q

What nucleotide bases does RNA contain?

A
  • A- adenine
  • G- Guanine
  • C-Cytosine
  • U- uracil
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8
Q

What base pairs pair together in RNA?

A

U pairs with A

C pairs with G

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9
Q

Even though RNA is single stranded, it can fold into specific structures. What does this involve?

What does this allow?

A
  • base pairing and covalent bonds

This allows some RNA molecules to have structural and catalytic functions

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10
Q

What types of RNA are there?

A
  • rRNA: Ribosomal RNA, form the basic structure of the ribosome and catalyse protein synthesis
  • tRNA: Transfer RNAs, central to protein synthesis as adaptors between mRNA and amino acids
  • Non-coding RNA, e.g. microRNAs, long non-coding RNAs
  • mRNA: Messenger RNA, codes for proteins (3-5%)
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11
Q

How DNA encodes for proteins in eukaryotes?

A
  1. Regulation of transcription is the most common form of control of protein production
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12
Q

What is a gene?

A

Entire nucleotide sequence required to direct protein synthesis

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13
Q

The structure of RNA polymerase in prokaryotes and eukaryotes

A

So, if you look at the crystal structure of rna polymerse either from a bacteria or from a yeast which is a eukaryotic organsisnm and what u can see is the 3 dimentional struactures of rna pol r very similar in both pro and euk cells and this underlies that the mechanism is very similar

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14
Q

What does RNA polymerase do?

A

Seperates the 2 strands of DNA

Uses one of the DNA strands as a template for RNA synthesis

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15
Q

Does RNA polymerase need a primer?

A

yes

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16
Q

Is RNA polymerase accurate?

A

Very accurate 1/10,000 bases

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17
Q

What direction does RNA polymerase move in?

A

5’ to 3’ direction

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18
Q

What does RNA polymerase synthesise?

A

A complementary RNA copy of the DNA template strand

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19
Q

How does RNA polymerase work?

A
  • well what the enzyme does is simply melt apart the 2 strands of DNA
  • It then uses 1 of the DNA strands as a template for rna synthesis
  • And it doesn’t require a primer
  • So up till now u have been talking a lot about DNA polymerases which require a primer – rna pol does not require a primer
  • And its’ fairly accurate making a mistake only once in every 10000 bases
  • And if u think about it mistakes in transcription aren’t so important as a mistake in DNA replication because what we r making is rna which has a very short half life
  • So even if the mistake was 2 be made in the rna thee would be a new rna which is going 2 come along and replace it very soon
  • So, mistakes in rna aren’t that important because rna is a transient entity with a really short half-life and will soon be replaced by another rna
  • So, there isn’t proof reading at transcription
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20
Q

Are there multiple RNA polymerases per gene?

A

yes

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21
Q

Are their multiple transcripts per gene?

A

Yes

As soon as the first RNA polymerase starts to move down the gene, the next polymerase can bind and initiate RNA transcription

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22
Q

What are the stages of transcription?

A
  1. Initiation
  2. Elongation
  3. Termination
  4. Processing
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23
Q

Transcription

A
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24
Q

Whats the first step to transcription?

A
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25
Q

Whats the second step to transcription?

A
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26
Q

Whats the third step to transcription?

A
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27
Q

Transcription is similar in prokaryotes and eukaryotes, however what are some of the differences?

A
  • initiation
  • promotor
  • RNA polymerase
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28
Q

Control of transcription is the major way protein expression is controlled. how is protein synthesis increased and decreased?

A
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29
Q

Why is the ability to control the level of transcription key?

A
  • Size of baby determined by placenta, looking at AA in food source and the transport of them, so more AA and more proteins would be more goes to the baby resulting in a larger sized baby
  • These steps are exactly the same in a human cell and in a bacteria cell
  • What is also similar between bac and hum cell is that the control of transcription is a major way in which protein expression is controlled
  • So, if a bac cell or a hum cell wants more of a particular protein wot it will do is increase the transcription of that gene, so u get more rna and therefore more protein
  • So, if a cell regardless of wot it is wants more of a protein will increase the rate of transcription, so u get more rna and more protein
  • Conversely if u want less of a particular protein transcription rate decreases and u get, less rna and therefore less protein
  • So, this ability to control the level of transcription from a gene is absolutely key
  • So, we will start by looking at how transcription is controlled in bacterial cells
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30
Q

Why is control of transcription so important?

A

can lead to cell death

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31
Q

Protein over-expression in E. coli triggers adaptation analogous to antimicrobial resistance

A
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32
Q

Summary to lecture 1

A
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33
Q

Are the steps to increase or decrease protein synthesis the same in human and bacteria cells?

A

Yes

  • So, if a bac cell or a hum cell wants more of a particular protein what it will do is increase the transcription of that gene, so u get more rna and therefore more protein
  • So, if a cell regardless of what it is wants more of a protein will increase the rate of transcription, so u get more rna and more protein
  • Conversely if u want less of a particular protein transcription rate decreases and u get, less rna and therefore less protein
  • So, this ability to control the level of transcription from a gene is absolutely key
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34
Q

Whats the difference between Constitutive genes and regulated genes?

A

Constitutive genes: always on, needed all of the time

Regulated genes: depends on environment and food source

35
Q

Tell me some examples of how regulated genes are affected by food source and environment

A
  • e.g. changes in food sources

Switches on genes that encode enzymes which are needed to metabolise that sugar. Wants glucose as this is the most useful for it

  • e.g. changes in environmental stresses (pH and temp)

Switches on genes that encode proteins which help the bacterium survive

36
Q

Whats an Operon?

A

Genes encoding for proteins in the same pathway are located adjacent to one another and controlled as a single unit that is transcribed into a polycistronic RNA (mRNA that encodes several proteins and is characteristic in many bacteria and chloroplast mRNA)

They also have no introns

37
Q

Whats the Lac operon?

What do the operon enzymes do?

A

It modifies its function for changes in lactose concentration seen

It switches on genes that encode enzymes which are needed to metabolise that sugar

Beta galactosidase: Lactose –> Glucose and galactose

Permease: Allows lactose to enter the cell

Transacetylase: An enzyme that transfers an acetyl group from acetyl-CoA to galactosides, glycosides and lactosides

38
Q

Where does the control of transcription most commonly occur?

A

At the initiation stage of transcription

39
Q

What are promoters the site of?

A

Transcription initiation in the DNA

40
Q

Tell me about promoters, their start/stop points etc.

A
  • Theres no 0, so upstream of +1 is -1, -2 etc.
41
Q

What are promoters recognised by and what do they notice?

A

Promoters are recognised by RNA polymerase by have a consensus (common pattern of) DNA sequence

42
Q

What different sequences are found in promoters?

What is their location?

A
  • hexamer (6bp) at -35 and a TATAAT sequence at -10
  • asymmetric (only in 1 DNA strand), hence RNA polymerase knows which way to go
43
Q

What defines a consensus sequence?

A

Its defined by aligning all known examples as to maximise homology

44
Q

Whats the purpose of Down-mutations and up-mutations?

A

Down-mutations: decrease the promoter efficiency, it usually decreases conformance to the consensus sequence

Up-mutations: increase the promoter efficiency, usually increases conformance to the consensus sequence

45
Q

Down mutation example 1

46
Q

Down mutation example 2

47
Q

What are the classes of bacterial promoters?

48
Q

What is a holoenzyme consisted of?

Whats the role of the subunits?

A

A Holoenzyme (complete enzyme) consists of:

  • 4 types of subunit
  • 5 subunits in total:

2x alpha subunits (40 kD) – enzyme assembly

beta, beta’ (prime) = form catalytic centre

σ and sigma (70 kD) – binds promote

49
Q

What subunits of the holoenzyme make up the core enzyme?

Tell me about the core enzyme

A

alpha, beta and beta’

The core enzyme has a general affinity for DNA- this is known as loose binding

It scans DNA and has a low affinity for DNA and looks out for a promoter site

50
Q

Why is the holoenzyme have affinity for DNA?

A

Positiviely charged Mg2+ and Zn2+ bound ions which has affinity for the negatively charged DNA

51
Q

What does the sigma unit ensure?

Tell me about the sigma unit?

A

The sigma unit ensures RNA polymerase only binds at promoter sequences

  • Increases affinity by about 1000 X binding strength
  • There is enough σ unit for 1/3 of polymerases hence other sigma factors are present and these subunits can be swapped in and out for specificity
52
Q

Tell me the use of alternative sigma factors

A
  • e.g. Heat Shock
  • σ70 is known as the house keeping factor
  • σ32 is induced by high temperatures
  • σ 32 is induced by the accumulation of unfolded proteins
  • σ 32 recognises a different -35 and -10 sequence
53
Q

Transcription summary in prokaryotes

A

Initiation: RNA polymerase binds to the promoter

  • consensus DNA sequence
  • regulates transcription

Elongation: RNA polymerase makes an RNA copy complementary to the template strand

  • Bacterial polymerase: only 1, with 4 units (2a, b, b’, σ)
  • Operons, no introns, no post-transcriptional modification of RNA

Termination: RNA polymerase stops when it recognises termination sequences (prokaryotes)

54
Q

Tell me about the initiation of transcription in bacteria

A
  • main regulatory step of transcription
  • RNA polymerase binds to the promotor
  • Unwinds the DNA
  • Transcription begins
55
Q

Tell me about the elongation stage of transcription in bacteria?

A
  • RNA polymerase makes an RNA copy complementary to the template strand
  • Bacterial RNA polymerase: only 1, with 4 units (sigma)
  • Doesn’t contain the sigma factor as it is released
56
Q

Tell me about the termination stage of transcription in bacteria

A
  • RNA polymerase stops at a termination sequence
  • RNA polymerase
  • RNA released
57
Q

What is transcription controlled by?

A

Regulatory proteins (transcription factors)

58
Q

Whats an example of negative regulation?

A

Transcriptional repressors

59
Q

Whats an example of positive regulation?

A

Transcription activators

60
Q

What is the lac operon regulated by?

A

Both the negative and positive regulators

61
Q

Tell me about regulation of the lac operon by the lac repressor when lactose is absent and present

A

Lactose absent

  • Lac repressor is produced
  • Lac repressor binds to the operator
  • Lac operon transcription blocked

Lactose present

  • Allolactose is produced (modification of lactose)
  • Allolactose binds to the lac repressor
  • This induces a conformational change in LacI
  • Repressor cannot bind to operator
62
Q

Which site does the lac repressor bind to?

Whats a palindrome sequence?

A

The operator site at the +1 region

It overlaps with the start site of transcription

Palindrome: DNA sequence that reads the same on each strand when the strands are read 5’ to 3’

63
Q

Lac operon in the absence of lactose

64
Q

Lac operon in the presence of lactose

65
Q

Tell me about the structure of the lac repressor?

A
  • A tetramer
  • Contains 2 dimers
  • Each dimer within the tetramer can bind to one operator site
66
Q

How many operator sites does the lac operon have?

67
Q

What operator sites can the lac repressor bind to and which ones allow repression/ no repression?

A
  • Knock out different sites to look at repressions
  • The lac repressor must bind 2 out of 3 sites
  • repressor can bind to O1 and O2 or O1 and O3
  • But not to O2 and O3
68
Q

When the lac repressor binds, this are the tetramer shapes produced

69
Q

What experiment proved that the lac repressor binds to 2 operator sites?

A

1996, X-ray crystallography

70
Q

Some promoters are only poorly recognised by RNA polymerase. Which means they have a low transcription rate, what can these promoters be enhanced by and provide an example

A

They can be enhanced by transcriptional activators

e.g. CAP (Catabolite Activator Protein)

71
Q

What does CAP use to judge levels of glucose?

72
Q

How does CAP function in High lactose- Low glucose scenarios?

A
  • strong transcription of the lac operon occurs.
  • The lac repressor is released from the operator because the inducer (allolactose) is present.
  • cAMP levels are high because glucose is absent, so CAP is active and bound to the DNA.
  • CAP helps RNA polymerase bind ot the promotor, permitting high levels of transcription.
73
Q

How does CAP function in levels of High lactose- High glucose?

A

(wants to use glucose at this point as prefers glucose over lactose):

Low-level transcription of the lac operon occurs.

The Lac repressor is released from the operator because the inducer (allolactose) is present.

cAMP levels, however, are low because glucose is present.

Thus, CAP remains inactive and cannot bind to DNA, so transcription occurs at a low, leaky level

74
Q

How does CAP function in low lactose and low glucose levels?

A

No transcription of the lac operon occurs.

cAMP levels are high because glucose levels are low, so CAP is active and will be bound to the DNA

However, the lac repressor will also be bound to the operator (due to the absence of allolactose), acting as a road block to RNA polymerase and preventing transcription

75
Q

How does CAP function in low lactose and high glucose levels?

A
  • when glucose levels are low, cAMP levels are high
76
Q

CAP and lac repression

77
Q

Tell me about the intrinsic terminators that prokaryotes have?

A
  • Termination sequence in the DNA
  • intrinsic terminators are a run of ATs in the template strand
  • roughly 50% genes
  • AT makes double helix looser if there are more of them and vice versa for GC due to the H bonding thats present
78
Q

Whats the stem-loop structure in RNA formed by?

A

Inverted repeats

e.g. CCCCXXXGGGG

79
Q

What does the stem-loop structure cause RNA to do?

What happens when this occurs?

A

pause

  • When the Polymerase pauses the RNA: DNA hybrid unravels from the weakly bonded A: U terminal region
  • The sequence of the hairpin and length of the U rich region determine the efficiency of termination (2 -90%)
80
Q

What is Rho?

Tell me about its structure?

How does it move?

A
  • A prokaryotic transcription protein
  • roughly 275kD hexamer, has a ring like structure
  • Each subunit has an RNA binding domain & an ATP hydrolysis domain moves along the RNA
  • Moves at same pace as RNA polymerase as it moves along DNA
  • RNA slows down at loop and rho starts to catch up and unravel connection
  • Rho dependent and independent termination process
81
Q

What does Rho dependent termination require?

A

An inverted repeat, stalls the RNA polymerase

82
Q

As Rho is a helicase what does this mean?

A

Unwinds DNA: RNA hybrids

83
Q

Bacterial transcription control summary

A

Transcription initiation controlled by:

  • Repressors
  • Activators
  • Lac operon controlled by both

Lac repressor: binds to 2 operator sites

  • Low lactose

CAP activator: helps RNA polymerase bind to promoter

  • Low glucose, signalled by cAMP

Termination: RNA polymerase stops when it recognises termination sequences

  • A-Ts leading to a stem loop
  • Rho dependant

BIOL2010 (7 decks)

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