Prokaryotic Transcription And Control Of Gene Expression

Question 1

What is the size of E.coli genome and how many genes

Answer 1

~4300 genes, 5Mb genome

Question 2

How many proteins are made at a time in E.coli and what type are made at high and low levels

Answer 2

~3000
High levels= ribosomal proteins (10,000/cell)
Low levels= regulators of transcription (<10/cell)

Question 3

What are the two classes of genes

Answer 3

Housekeeping/ constitutive- always expressed, amount not highly regulated
Inducible- expression is induced or repressed, highly regulated

Question 4

What are the reasons why gene expression is regulated

Answer 4

To conserve resources
Respond to changes in the internal/ external environments (nutrients, building blocks, toxins)
Ordered development

Question 5

How are translation and transcription coupled. What speed do they occur at and why are they coupled (only occurs in prokaryotes as they have no nucleus)

Answer 5

mRNAs are rapidly degraded (half-life is 2-3min) so transcription and translation are coupled and occur quickly to prevent mRNA degradation
Transcription= 40N/s
Translation= 15 aa/s (similar to above)

Question 6

Which DNA stand is the coding strand and which is the template strand

Answer 6

5-3’ is coding

3-5’ is template= transcribed into mRNA

Question 7

What enzyme transcribes DNA into RNA in prokaryotes

Answer 7

RNA polymerase- DNA dependent RNA polymerase

Question 8

What is an operon

Answer 8

Set of co-transcribed genes under the control of a single promoter

Question 9

Why are genes in operons co-regulated and what are their transcripts

Answer 9

Genes usually have related functions so can be co-regulated. Can also have unknown functions. Transcripts are polycistronic meaning multiple genes/ cistrons encode multiple proteins

Question 10

What are non-coding RNA (give examples) and how do they function

Answer 10

Dont code for proteins but may be processed
Eg tRNA, ribosomal RNA, CRISPR RNA
Can function alone or as RNA-protein complexes like ribosomes
Often bind specifically to other RNAs or DNA by base pairing eg mRNA codon- tRNA anticodon

Question 11

What does CRISPR stand for

Answer 11

Clusters of regulatory interspaced short palindromic repeats

Question 12

What are the two parts to CRISPR

Answer 12

Immunisation process (adaptation) and interference process

Question 13

How does the immunisation process in CRISPR work

Answer 13

foreign DNA inserted
CRISPR Cas proteins expressed from cas operon which recognise foreign DNA and cleave/ cut out a piece
Curt out DNA is inserted into gene encoding for CRISPR ncRNA leading to tags of foreign DNA with common pieces/ repeats between them

Question 14

How does the interference process of CRISPR work

Answer 14

CRISPR repeat spaced ncRNA transcribed into pre-crRNA which is processed into mature crRNA
crRNA combine with cas proteins and used as guide by cas complex to inactivate corresponding nucleic acid

Question 15

What are the three steps of transcription

Answer 15

Initiation, elongation, termination

Question 16

What is a promoter region and how are they recognised

Answer 16

Where RNA polymerase binds (alpha helicies of sigma factor bind to DNA major groove)

Question 17

What is a sigma factor and how do they allow recognition of different promotor regions

Answer 17

Protein subunit of RNA polymerase. There are alternative types which under different conditions, recognise different promotor sequences. They coordinate distantly located operons

Question 18

What are the different subunits of RNA polymerase (RNAP) holoenzyme

Answer 18

Beta subunits which are catalytic
Sigma which provide specific binding to DNA
Alpha subunits which are for enzyme assembly, promotor recognition and bind some activators
aaBB’ is the core enzyme, sigma provides specificity
Also w in some RNAP so can be aaBB’w

Question 19

Steps in transcription initiation and elongation

Answer 19

Non-specific binding of RNAP, moves to promotor
Sigma binds when promotor is recognised and causes closed complex
Open-promotor complex formed where strands are separated
Initiation of mRNA synthesis- almost always with a purine
Initiation can be abortive- releasing 2-9 oligonucleotide chains)
Elongation of non-abortive mRNA
Release of sigma as polymerase proceeds down template away from promotor

Question 20

What are the coding and non-coding strands in transcription of prokaryotes

Answer 20

Sense (coding) strand

Antisense (noncoding/ template) strand

Question 21

What is Rifampicin (RIF) and how does it work

Answer 21

Transcription inhibitor of tuberculosis protein. Resistance to this can arise
Binds to catalytic beta subunit of RNAP and stalls transcription (used with two or three other drugs for 6 months)
If resistance occurs, other drugs must be used instead

Question 22

What are the two methods of transcription termination and a brief overview

Answer 22

Rho independent- most common, specific hairpin structure in RNA formed from complementary base pairing in the RNA and the poly-A tail creating a poly-U tail in RNA at the end
Rho dependent- Rho protein (hexomer) binds to RNA and stops transcription, is ATP dependent also

Question 23

Features of RNA that differ to DNA and make it able to form a hairpin for transcription termination

Answer 23

RNA helix has hole down the middle
RNA has narrower and deeper major groove
Extra OH group
Same number of bonds between bases when they pair (A=U and C=-G)
Bases at the top of the loop (actual round part) do not pair together

Question 24

What things does the regulation of transcription depend of

Answer 24

Promoter strength- degree of match, weak or strong for sigma factor
Presence of alternative sigma factors active under different conditions
Presence of transcription factors via positive or negative regulation (binding affinity of these can be modified allosterically or covalently

Question 25

What is positive and negative regulation of the presence of transcription factors

Answer 25

Negative regulation- there is an inhibitor present which stops constant transcription and an inducer removes the inhibitor when transcription is required
Positive regulation- an activator binds to the DNA when transcription is required

Question 26

Briefly, how is the lac operon transcription regulated

Answer 26

When glucose is low and the cell has lactose available, lactose is converted into allolactose. Allolactose acts as an inducer for the lac operon repressor (which is made from I mRNA found on the gene)
Requires inducer (+ lactose and therefore + allolactose) and low glucose
When there is low glucose, ATP is converted into cAMP which is collected by cAMP receptor protein (CRP) and binds to DNA allowing RNA polymerase to bind and undergo transcription

Question 27

What are the three parts of the lac operon and what do they do

Answer 27

Z= B-galactosidase which works to convert lactose to glucose and galactose
Y= permease which transports lactose into cells
A=trans-acetylase

Question 28

Why is it important that a small amount of expression occurs when the lac operon is repressed off?

Answer 28

Because a small amount of permease is needed to get the lactose into the cell
Because a small amount of betagalactosidase is needed to produce the inducer (allolactase)

Question 29

Features of the trp operon

Answer 29

Biosynthetic operon
Order of genes is the same as the pathway (enzymes made in order of what is needed)
Enzymes arent made if tryptophan is already available

Question 30

How does the trp operon feedback loop one work

Answer 30

Trp binds trpR and corepresses operon. TrpR is repressor and tryptophan is corepressor
TrpR gene becomes repressor mRNA and then the trp repressor. Binds with trp, changes conformation and binds to the promoter and operator regions to block transcription. When the repressor and corepressor is taken away transcription can occur.
The making of repressor mRNA is an ongoing but slow process

Question 31

How does the trp operon feedback loop two work

Answer 31

Trp operon regulated by attenuation= regulated transcription termination
When tryptophan levels are high, only the attenuated sequence is transcripted but when tryptophan levels are low the whole trp mRNA is transcribed

Question 32

Brief description of how attenuation works in trp operon

Answer 32

TrpL mRNA can form alternative RNA structures
When tryptophan abundant, forms a terminator early in mRNA so no trp operon synthesis occurs. Ribosome goes to gene 2 to stop it from base pairing with gene 3. Then genes 3 and 4 base pair and form a transcription terminator
When tryptophan low, ribosome stalls at tandem trp codons on gene 1 allowing genes 2 and 3 to base pair and this doesnt form a transcription terminator, meaning transcription continues
Requires coupling of transcription and translation

Question 33

How can bacteria be forced to produce large amounts of tryptophan

Answer 33

Removing feebback
Selected bacteria that made high levels of tryptophan, duplicated all trp genes, replaced trp promoter and leader with strong unregulated promoter and added extra copies of genes for proteins that make precursors