Prokaryotic Genetics

Question 1

Where is prokaryotic DNA found

Answer 1

Nucleoid

Question 2

What are the main similarities between eukaryotic and prokaryotic DNA replication

Answer 2

Both start at the origin
Bidirectional
Semiconservative

Question 3

What does the bacterial origin contain (base pairs)

Answer 3

AT Rich 13bp and 9bp sequence

Question 4

How does DNA replication in prokaryotes begin

Answer 4

An initiator protein DnaA binds to 9-bp repeat regions in the origin
- which leads to the denaturation of a13-bp region

Question 5

What happens once denaturation occurs at the 13bp region

Answer 5

DNA helicase DnaB is recruited and loaded onto DNA by the helicase
loader DnaC. Helicases untwist DNA in both directions.

DNA primase DnaG is recruited and
synthesises an RNA primer

Question 6

What happens once the RNA primer has been synthesised in bacterial DNA replication

Answer 6

DNA strands are synthesised by DNA
polymerase III
DNA pol III is a holoenzyme made up of numerous subunits for various
functions e.g. DnaN (DNA clamps keep the enzyme bound to DNA),
DnaQ (3’-5’ exonuclease activity) or holE (proofreading activity

Question 7

What is the role of terminator sequences

Answer 7

To end replication

Question 8

How does the terminator sequence work

Answer 8

Tus binds to terminator sequences and only
allows replication forks to proceed from one
direction
Replication forks stop on the opposite side of the origin
Two daughter molecules are then separated by topoisomerase IV and breaks are sealed
by DNA ligase

Question 9

What is the role of DNA pol III

Answer 9

Elongation

Question 11

What is the role of helicase in bacterial DNA replication

Answer 11

Unzips the double helix

Question 12

What is the role of lipase in bacterial DNA replication

Answer 12

Seals the gaps between the Okazaki fragments on the backbone of the lagging strand
to create one continuous DNA strand

Question 13

What is the role of primase in bacterial DNA replication

Answer 13

Synthesises RNA primers needed to start replication

Question 14

What is the role of single-stranded binding proteins in bacterial DNA replication

Answer 14

Bind to single stranded DNA to prevent hydrogen bonding between DNA strands, reforming double stranded DNA

Question 15

What is the role of DNA gyrase in bacterial DNA replication

Answer 15

Relaxes supercoiled chromosome to make DNA more accessible for the initiation of
replication; helps relieve the stress on DNA when unwinding, by causing breaks and
then resealing the DNA

Question 16

What is the role of DNA topoisomers IV in bacterial DNA replication

Answer 16

Introduces single-stranded break into concatenated chromosomes to release them
from each other, and then reseals the DNA

Question 17

How is DNA packaged to Fit inside the cell

Answer 17

Supercoiling

Question 18

What is a negative supercoil

Answer 18

Is DNA is twisted in the opposite direction of the double helix

Question 19

What is a positive supercoil

Answer 19

If DNA is twisted in the same direction as the double helix

Question 20

What bacterial proteins are involved in maintaining super coils

Answer 20

• HU and Integration host factor (IHF) binds to specific sequences and introduces sharp bends in DNA

-Topoisomerase I and DNA gyrase maintain supercoil

Question 21

For initiation to commence…

Answer 21

A polypeptide called sigma factors bind to form a holoenzyme

Question 22

What does the sigma factor prevent

Answer 22

RNA polymerase binding to unspecific
sequences and to stably bind to -10 and -35 promoter
sequences

Question 23

Describe the steps in elongation in bacterial DNA replication.

Answer 23

Initial binding between polymerase and promoter is referred to as a closed
promoter complex as the DNA is not unwound

RNA polymerase unwinds ~15 base pairs to form an open promoter
complex where single stranded DNA is now available to be used as
a template

After adding around ~10 nucleotides,
the sigma factor is released from the
holoenzyme

• RNA polymerase leaves the promoter and the transcription bubble along template DNA to continue elongation

• DNA behind RNA polymerase reform into
double-stranded DNA

Question 24

What is a rho -independent terminator sequence and how does it work

Answer 24

consist of an inverted repeat sequence followed by a string of adenines

The inverted repeat region allows
the RNA to form a hairpin loop
structure

The hairpin causes RNA polymerase
to slow and then pause

Uracil chain destabilises pairing between new RNA and
DNA strand, causing RNA polymerase to dissociate

Question 25

What is a rho -dependent terminator sequence and how does it work

Answer 25

don’t use an inverted hairpin loop Rho-dependent terminator sequences contain a binding site for Rho protein to bind Rho moves along the transcript until it reaches RNA polymerase Rho is a helicase which unwinds the RNA-DNA helix, resulting in release of new RNA strand and RNA polymerase

Question 26

Describe the initiation steps for translation in prokaryotes

Answer 26

IF-3 and IF-1 initiation factors bind to the small 30S subunit The 30S subunit then binds to the mRNA upstream of the AUG start codon at the Shine-Dalgarno sequence An initiator tRNA binds to the AUG start codon carrying a modified form of methionine called formylmethionine Binding is facilitated by the IF-2 initiation factor The 50S subunit binds leading to release of the 3 initiation factor

Question 27

What are the 3 release factors in bacteria and what do they recognise

Answer 27

Rf1 recognises UAA and UAG Rf2 recognises UAA and UGA Rf3 dissociates RF1 and RF2 from stop codons. GTP hydrolysis facilitates RF3 dissociation

Question 28

What are operons

Answer 28

The groups bacterial genes are organised into are clusters of coregulated genes that are under the control of a single promoter • Genes in operons are switched on or switched off together

Question 29

What does grouping genes allow bacteria to do

Answer 29

allows bacteria to adapt to rapidly changing environments e.g. change in energy sources

Question 30

Describe the stages of the control of prokaryotic gene expression

Answer 30

Repressors bind to sequences called operators which block RNA polymerase, preventing it transcribing genes Activators are regulatory proteins which increase transcription of operons by facilitating RNA polymerase binding to the promoter Small molecules can also influence operons indirectly by binding to regulatory proteins and switching its conformation – allow or prevent binding to operon