Gene Structure and Bacterial Transcription

Question 1

Intron vs exon?

Answer 1

Intron = non coding sequence and allows for alternative splicing.
Exon = protein coding region

Question 2

What defines a gene?

Answer 2

A DNA sequence that is the template that encodes information for a functional product made of RNA or protein.
Only 25% of the genome meets this criteria.

Question 3

Is non coding RNA still a gene?

Answer 3

Yes: for example tRNAs and rRNAs still have vital functions

Question 4

Why must gene expression be highly controlled?

Answer 4

Gene expression control is essential for
-cells being able to move through the cell cycle
-cells responding to the external environment
-cells differentiating into different cells.
Not all genes need to be expressed at the same time or to the same degree -> gene expression is highly regulated from transcription through to translation.
As a general rule the amount of RNA transcripts is proportional to the number of proteins.

Question 5

How is transcription regulated?(eukaryotes)

Answer 5

Segments of DNA around a gene regulate gene expression via regulating the activity of RNA polymerase II which is responsible for creating the RNA molecule

Question 6

In bacteria: what are the main regulatory structures of the gene.

Answer 6

Promoter region
Transcriptional start site
Transcriptional terminator site
Note that these are different to start/stop codons involved in protein synthesis / translation.

Question 7

What does the promoter region do? How?

Answer 7

Sequence of DNA where RNA polymerase binds to synthesize the RNA transcript. These sequences are found before the gene and also direct the RNA polymerase to which DNA strand is to be used as a template.

Question 8

What are consensus sequences in a promoter region.

Answer 8

Consensus sequences in a promoter region of DNA refer to the specific nucleotide sequences that are recognized by transcription factors and RNA polymerase during the initiation of transcription. These sequences are found upstream of the transcription start site and play a crucial role in regulating gene expression by facilitating the binding of transcriptional machinery.

Question 9

Where are bacterial consensus sequences found and what are they?

Answer 9

TTGACA found at -35 and TATATT (Prinbow box) found at -10. These sequences have been shown to be conserved (in E.coli at least).

Question 10

What is a UP element?

Answer 10

Also known as an up stream element, UP elements are sequences rich in thymine and help strengthen the promoter region (can be found upstream of promoter region). These are not common to all promoter regions.

Question 11

What can deviations from consensus sequences cause?

Answer 11

Impaired binding of RNA polymerase to the promoter and affected gene expression.

Question 12

What are operator sequences and activator sequences

Answer 12

Operator sequences are specific to prokaryotes and are found either within the promoter regions (e.g. between the consensus sequences) or between the promoter region and the gene. They are binding sequences on which repressor proteins can bind and physically stop RNA polymerase from binding.
Activator sequences bind proteins that increase the efficiency of RNA polymerase binding to DNA by stabilising RNA polymerase. Activator sequences are especially important if there are mutations in the promoter region which make it hard for RNA polymerase to bind.

Question 13

What does a terminator sequence do and how? (prokaryote)

Answer 13

Stops the RNA from transcribing. This consists of consensus sequences cause the formation of a hairpin stalling the RNA polymerase. Hairpin: two nearby consensus sequences on the same strand that are complementary to each (e.g. TATA-> ATAT) bind to each other

Question 14

What is different about bacterial genes?

Answer 14

They are distinct and don’t overlap with one another
They can be polycistronic
They don’t have introns and exons

Question 15

What does monocistronic and polycistronic mean?

Answer 15

Monocistronic: 1 gene between a promoter and terminator sequence -> makes one mRNA transcript and protein
Polycistronic: multiple genes between a promoter and terminator sequence -> makes one mRNA transcript but can be used to make multiple proteins.

Question 16

How are polycistronic genes expressed?

Answer 16

A coordinated expression of genes in one pathway and often occurs by enzymatic pathways. All the different genes within the produced mRNA are regulated by the same promoter.

Question 17

What is the TATA box?

Answer 17

(TATAAAA - eukaryotic equivalent of the prinbow box) This is found in the promoter region of eukaryotes and is a common consensus sequence of type II genes and is where the TATA protein binds which helps RNA polymerase II bind to initiate transcription. Typically found 25 base pairs from the start site. RNA polymerase II is the polymerase used in eukaryotes for coding mRNA transcripts, I and III do other stuff.

Question 18

What are the common upstream elements that can be found in eukaryotes? Where are they found and what do they do?

Answer 18

CAAT box (GGCCAATCT) and GC box (GGGCGG)
These consensus sequences can be found 50 to 200 base pairs from the start site.
Most eukaryotes have one of these and they are binding sites for transcription factors (proteins that help RNA polymerase bind). These are found upstream from the TATA box btw.

Question 19

What are regulators and what is their role?

Answer 19

The eukaryotic equivalent of operators. They can either act as enhancers or silencers of gene expression. This is crucial for ensuring the right genes are expressed when needed in the appropriate cells. Silencers and enhancers can sometimes occupy the same space competing with one another to regulate gene expression.

Question 20

How do regulators affect gene expression over long distances?

Answer 20

Eukaryotic equivalent of operators/activators but are called enhancers or silencers and they can compete with each to affect gene expression and are sometimes in the same location. The upstream control elements bind general transcription factors which recruit the mediator complex which can then interact with the proteins binding to the regulators significantly further upstream along the DNA. Unlike prokaryotic operators, regulators can be very far from the start site. This works as DNA can be orientated in such away that distant sequences can come into close proximity to each other by the function of specialized proteins that can hold eukaryotic chromosomal DNA in loops, which increases the association of a gene with a regulator like an enhancer or repressor.

Question 21

Where generally on a chromosome do you find heterochromatin and euchromatin. What does this imply?

Answer 21

Heterochromatin: telomere and centrosome as there is high quantity of chromatin and must be condensed.
Euchromatin: everywhere else.
Genes are found in the euchromatic region as it would inefficient/damaging to condense and decondense the heterochromatic areas for gene expression.

Question 22

What do insulators do?

Answer 22

They have a barrier function stopping the spread of heterochromatin into euchromatic regions and stop other genes/chromatin structures interfering with another gene being expressed.
Insulators stop enhancers from interacting with the promoter regions of neighbouring genes ensuring they act on the promoter regions within their regulatory domain.

Question 23

Explain introns.

Answer 23

Intros are the non coding region between introns. These regions play important regulatory roles.

Question 24

What do eukaryotic regulations ensure?

Answer 24

Genes are expressed:
-at the right level
-in the right cell
-in response to the right conditions
-at the right time.

Question 25

What is a sigma factor?

Answer 25

A polymerase subunit that recognises and binds to promoter sequences. When the sigma factor and RNA polymerase combine they become the holoenzyme. The sigma factor is the part of the complex that attaches the RNA polymerase to the DNA.
Sigma factors are unique to prokaryotes.
There is variety of sigma factors for different genes one of the most common being no. 70 which is used for the expression of housekeeping genes.

Question 26

How does the holoenzyme prepare for transcription?

Answer 26

The sigma factor within the holoenzyme is responsible for separating or ‘melting’ the DNA strands. The sigma factor binds to only one strand of the double helix via the sigma factors corresponding consensus sequences and this determines which strand will be transcribed. Following binding the sigma factor melts the DNA.

Question 27

How is transcription initiated?

Answer 27

With the DNA ‘melted’ and the strands exposed transcription can begin. Ribonucleotides are added (thymine replaced with uracil) in 5’ -> 3’.
As transcription starts the sigma factor can dissociate and bind to another RNA polymerase.

Question 28

Explain how termination works.

Answer 28

RNA polymerase transcribes through the sequence. The hairpin is formed on the RNA strand by secondary structure forming due to the termination sequence and it causes the RNA polymerase to stall. This combined with a stretch of uracils following the hairpin means DNA and RNA start to dissociate from each other.

Question 29

Where in prokaryotes does translation and transcription occur? What does this mean?

Answer 29

Both occur in the cytoplasm. Proteins (namely ribosomes) are readily accessible in the cytoplasm and therefore new transcripts are exposed to ribosomes and translated. This means that translation and transcription can occur simultaneously unlike in eukaryotes and RNAs aren’t modified.

Question 30

What are operons and their purpose?

Answer 30

Polycistronic genes that allow for coordinated expression of multiple genes under the control of one promoter.
They are important for:
-Being able to respond quickly to the environmental conditions is critical for bacteria to ensure that they can adapt as bacteria are more vulnerable to the environment.
-As they are single cell organisms, their need for tight transcription control is needed to ensure efficient use of energy and resources
-Operons allow for a coordinated response to achieve this

Question 31

What does lac operon do?

Answer 31

The different genes of the lac operon code the proteins involved in the metabolism of lactose into glucose and galactose. Common regulation of the gene makes it efficient for responding to stimulus e.g. cell energy requirements.

Question 32

Describe the baseline activity/regulation of the lac operon.

Answer 32

Its baseline state is turned off due to lac repressor protein. Lac repressor is coded by ‘lacl’ gene which is considered the trans regulator of Lac operon (it has its own promoter and terminator). The lac repressor is always transcribed and expressed. The repressor protein binds to the operator in the Lac operon preventing RNA polymerase from binding.

Question 33

Explain how allosteric regulation works.

Answer 33

Regulatory proteins sometimes need to undergo a conformational change so they are able to bind with DNA (in this context). Other molecules will bind to the allosteric site of these proteins creating the conformational change that exposes the DNA binding site. This creates a system of activation or inhibition that is utilised in response to stimuli.

Question 34

What are the different types of regulatory proteins and the effect of their interaction with an effector molecule?

Answer 34

Inducible repressors
-Do not inhibit transcription when bound to effector molecule (A)
Repressible repressors
-Inhibit transcription when bound to effector molecule (B)
Repressible activators
-Do not activate transcription when bound to effector molecule (C)
-Inducible activators
Cause transcription when bound to effector molecule (D)

Question 35

What is CAP? How and where does CAP bind to DNA?

Answer 35

cAMP-bound catabolite activator protein - CAP
When a cell runs out of glucose it produces cyclic AMP from breaking down ATP. (low glucose = high cyclic AMP) Cyclic AMP bound to CAP creates an inducible activator meaning CAP can bind to the DNA upstream of the Lac operon promoter. However if there is no lactose for the cell to metabolise, the repressor protein remains bound to the promoter stopping CAP from binding to DNA and recruiting RNA polymerase to the promoter.

Question 36

How is lac repressor removed?

Answer 36

If lactose is present some of it will be metabolised by another molecule into allolactose. This acts as an inducible repressor and binds to lac repressor protein causing a conformational change making it dissociate from DNA. This allows transcription of the lac operon to occur because the DNA bound CAP can recruit RNA polymerase to the promoter expressing the genes that allow lactose to be metabolised.

Question 37

How is lac operon returned to base line activity?

Answer 37

Cyclic AMP production reduces as more glucose is produced in the cell -> less CAP will bind to DNA. Eventually levels of allolactose will drop and lac repressor will bind to DNA again.
Dual system.