week 1

Question 1

How is DNA organised to fit into a prokaryotic cell?

Answer 1

Chromosome is compact due to DNA supercoiling

Question 2

Is DNA usually supercoiled positively or negatively? and why?

Answer 2

negatively to favour unwinding as local unwinding is needed for DNA recombination

Question 3

How is DNA organised in eukaryotes?

Answer 3

packaged tightly into chromatin

Question 4

How is chromatin stabilised?

Answer 4

stabilised by nucleosomes (DNA wrapped around histone)

Question 5

What is the definition of euchromatin and heterochromatin?

Answer 5

1. least condensed (rich in genes)
2. most condensed (very few genes)

Question 6

Many genes are arranged in clusters controlled by the same ___

Answer 6

promotor

Question 7

Explain lac operon when lactose is present vs absent

Answer 7

• when lactose is present, it binds to lac repressor protein and distorts its shape so it can no longer bind to operator region. RNA polymerase binds to promotor region and transcription of structural genes take place, producing lactase in the presense of lactose
• lactose is not present, the shape of the repressor protein is not distorted, therefore it binds onto the operator region blocking the RNA polymerase. As a result, the gene is switched off and no lactase is made

Question 8

What are plasmids?

Answer 8

pieces of DNA that contains non-essential genes, beneficial but not necessary

Question 9

In eukaryotes, each gene has their own p_____.

Answer 9

promotor

Question 10

eukaryotic genes contain exons and introns. what are they?

Answer 10

exons = coding regions
introns = non-coding regions

Question 11

What is the benefit of having introns?

Answer 11

alternative splicing of mRNA = different protein structures

Question 12

Describe the chromosomes in prokaryotes

Answer 12

• circular chromosome
• not membrane bond
• nucleiod = region of DNA with associated proteins

Question 13

Describe the chromosomes in eukaryotes

Answer 13

• linear chromosomes
• in pairs (homologues)
• 2 copies of each gene in each homologous chromosome

Question 14

Why are chromosomes more visible in prophase?

Answer 14

condensing of chromatin

Question 15

What is the chromosome territory?

Answer 15

chromosomes are arranged in a defined area of the nucleus.

Question 16

Compare sizes and density of genome prokaryote vs eukaryote

Answer 16

pro:
- small genome
- genes are close to each other
- high gene density

eu:
- large genome
- low gene density
- only 1% codes for proteins

Question 17

What are non-coding functional elements identified as?

Answer 17

• TF, microRNAs, interferring RNAs, long non coding RNAs

Question 18

What is syntheny?

Answer 18

comparing the organisation of human genome with other species can elucidate how species evolutionarily diverged. The genome of 2 species can be arranged into blocks where the order of genes is the same a their most recent ancestor.

Question 19

Why do most prokaryotic genes not contain introns?

Answer 19

• introns would inhibit translation coupled to transcription in prokaryotes
• introns appeared after eukaryotes diverged from prokaryotes

Question 20

What is the central dogma?

Answer 20

genetic can be transferred between RNA & DNA (transcription and reverse transcription) and to Protein (translation) but it can not be transferred from protein back to nucleic acids.

Question 21

Explain transcription

Answer 21

RNA polymerase synthesise RNA strand using DNA as a copy strand

Question 22

In bacteria, transcription and translation are ___

Answer 22

coupled

Question 23

Why is transcription/translation coupled in bacteria?

Answer 23

usually in eukaryotes transcription takes place in the nucleus and translation takes place in ribosomes in cytoplasm. however bacterias don’t have nucleus so ribosomes can translate whilst its still being transcribed

Question 24

Gene has x-axis: what is +1

Answer 24

First nucleotide of the transcript

Question 25

Gene has x-axis: what is -10 and -35

Answer 25

-10 = TATA box - they are important for RNA polymerase to recognise the promotor so they know where to bind and start transcribing

Question 26

- monocistronic mRNA: ____ - polycistronic (multigenic) ____

Answer 26

- one coding sequence = 1 protein - operons (one gene multiple proteins)

Question 27

prokaryotes have a single RNA polymerase core for all genes. explain the structure.

Answer 27

consists of 5 subunits, sigma factor has to join for active transcription. sigma factor required to recognise -10 and -35. sigma leaves when transcription starts bc it is no longer needed

Question 28

how does RNA polymerase know its in the transcriptional terminator?

Answer 28

short hairpin in the region of termination followed by a stretch of Us in the transcript. because the AU pair is weaker than CG, it causes the pairs to dissociate.

Question 29

There is a ____ equilibrium between occupied and unoccupied state of a promotor

Answer 29

dynamic

Question 30

Describe the equilibrium in a strong promoter setting and weak promotor setting.

Answer 30

equilibrium shifts towards the bound molecule. so more transcripts are made. in the case of a weak promotor situation, equilibirum shifts towards the promotor free situation. so less transcripts are made and less of the subsequent proteins are created.

Question 31

What are the 2 ways to compare promotor strength?

Answer 31

1. GFP 2. enzymatic reporter gene

Question 32

How to use GFP to compare reporter strength?

Answer 32

using fluorescent reporter gene coding for GFP (green fluorescent protein). if we fuse promotor 1 an d2 to GFP and look at cells, we will see that the brighter and fluorescent, the stronger the promotor. because stronger promotor → more GFP mRNA is made → more fluorescence protein → brighter colour

Question 33

How to use enzymatic reporter gene to compare reporter strength?

Answer 33

lacZ codes for B-galactosidase (enzyme) which catalyses the reaction where a di-sugar (no colour) is broken down into 2 mono-sugars (one being yellow). the more intense the reaction, the more yellow the colour = stronger promotor

Question 34

4 ways to change the amount of protein produced (altering gene expression)

Answer 34

1. transcriptional level = more RNA transcripts → more proteins 2. post-transcriptional level = more stable RNA transcripts → more proteins 3. translational level = premature translation termination → less protein 4. post-translational level = protein degration → less protein; activation or inactivation of a protein by covalently attatching phosphate, acetyl, sumo, ubiquitin and other groups (characteristics of eukaryote not prokaryotes

Question 34

How does alternative sigma factors activate specific sets of genes?

Answer 34

sigma factor tells the core enzyme which promotors to bind to in order to express certain genes The -10 and -35 boxes are recognised by sigmas and they have different sequences for different sigma factors.

Question 35

What is a regulon?

Answer 35

the genes that are triggered by different sigma factors are scattered throughout genome but are all grouped in regulon.

Question 36

sigma independent regulation using transcription factors. divided into 2 groups

Answer 36

repressors or activators

Question 37

Describe negative gene regulation using repressors

Answer 37

- transcription by default is on, if theres no transcription factor than transcription will take place - if transcription factor is in place and it codes for a repressor protein, then repressor can shut down transcription by binding at the promotor, physically blocking RNA polymerase from binding to promotor - repressor binds to operator region

Question 38

Describe positive gene regulation using activators

Answer 38

- RNA polymerase has a weak binding ability to promotor (hard to bind due to -10 and -35 regions that binds to sigma) - if an activator is in place, it binds on the promotor region (activator binding site) - it undergoes a reaction where the activator anchors the RNA polymerase at the promotor, thereby helping transcription

Question 39

Lac operon is weak even with repressor inactive so we require an activator called CAP. CAP can't bind unless _____ is present ____ is a regulatory molecule anti is ____ by glucose if glucose isn't present ____ is made and binds to CAP and CAP is bound to ____

Answer 39

cAMP cAMP inhibited cAMP activator binding site

Question 40

Both CAP and Lack is regulated by cAMP and lactose through conformational change of proteins. these effectors bind to ______ so this type of regulation is called ____

Answer 40

allosteric site allosteric regulation

Question 41

What is lactose made of?

Answer 41

glucose and galactose

Question 42

What causes lactose intolerance>

Answer 42

decreasing expression of the LCT gene. leads to sustained lactase production

Question 43

Describe the 3 levels of expression of the lactase gene

Answer 43

1. lac repressor bound 2. lac repressor not bound but activator not bound 3. lac repressor not bound activator anchors RNA polymerase

Question 44

What are transposons?

Answer 44

mobile elements which can jump from one DNA molecule to another (e.g. plasmid to chromosome), codes for antibiotic resistance genes

Question 45

What is horizontal gene transfer?

Answer 45

using transposons to migrate between bacteria and spread antibiotic resistance

Question 46

What are smaller plasmids used to construct?

Answer 46

clowning vectors used carry DNA fragments of interest

Question 47

Describe bacterial mating via conjugation

Answer 47

plasmid transfer coupled with DNA replication