Gene mechanisms

Question 1

Steps in Initiation pathway for DNA replication

Answer 1

1) Orc binds and then loads 2 other proteins – Cdt1 and Cdc6
2) Cdt1 interacts and loads the MCM proteins – final complex = preRC – pre-replication complex
3) Marked as a specific stage because it’s stable and can isolate the replication complex bound to DNA from cells
4) Waits and then is activated and then forms the pre-initiation complex – occurs by addition of proteins
5) Then elongation occurs after pre-initiation complex.

Question 2

What is the transition from initiation to elongation in DNA replication

Answer 2

• Transition from preRC to preIC = changes in post translation modification of proteins.
• There can be loading of additional new proteins that can be unloading of preRC and preIC proteins – converts complex to active replication fork – that can replicate bulk of genome

Question 3

Okazaki fragment initiation

Answer 3

1. Primase synthesises primer – extended by polymerase alpha – continues to synthesise till it meets with rpa on the template
2. Cause polymerase alpha to drop off and a 5-subunit enzyme = rfc – binds to primer terminus – loads pcna molecule onto DNA
3. Opens pcna ring and closing it around the DNA – pcna forms the polymerase clamp. Rfc referred to as clamp loader.
4. Pcna bound then loads polymerase delta – produces fragments that need to be joined together

Question 4

Steps in joining of the Okazaki fragments

Answer 4

1. RNA and some of the DNA synthesised by polymerase alpha is removed – 3 enzymes responsible: Rnaseh, Fen1, DNA2
2. Leaves a gap in template – filling in occurs by loading Rfc, Pcna, polymerases and the polymerase switch  nick in DNA
3. Nick joined together by ligase – DNA ligase 1

Question 5

What are the leading strand factors

Answer 5

Polymerase
Rpa
Pcna

Question 6

Function of Rpa

Answer 6

single stranded binding protein

• Binds to single stranded DNA that is revealed in unwinding – prevents it from being degraded

Question 7

Function of pcna

Answer 7

Processivity factor for the polymerase – Prokaryote version = DNAb
• Pcna with polymerase stays tethered = increase amount of time it stays on the DNA
• Prescence can show if cells are dividing or not
• Also have function in chromatin loading and repair – related to tethering proteins on to chromatin and coordinating DNA replication

Question 8

What does processivity mean

Answer 8

Amount of time the polymerase stays associated with the template in 1 go – number of nucleotides it synthesises before it dissociates

Question 9

Function of topoisomerase

Answer 9

during synthesise generate supercoils that must be removed – as replication is going on or when 2 forms collide and some of these are trapped between them and will need to be removed

Question 10

Spindle dynamics

Answer 10

• Spindle poles duplicate and the new spindle pole body moves around the edge – assisted by motors
• Spindle is generated – invades into region with chromosomes and searches for the kinetochores
• Microtubules also sent out -> cytoplasm to anchor the spindle so it can generate mechanical force it needs
• Both attached so pull from spindle pole are equal so stay in middle – all attached they line up along central region of nucleus = metaphase plate

Question 11

Structure of polymerase 2

Answer 11

• Contains a cleft – DNA enter the complex so that it can be transcribed
• DNA enters into cleft and JAWS grip the DNA – JAWS may open/close
• When DNA gets close to the active site – it’s bound by the bridge helix and ‘switch’ regions during elongation – stabilises the elongation complex.
• Linear DNA exit is blocked by the ‘wall’ and it exits at a 90o angles
• Nucleotides enter via a port below the active site.

Question 12

Steps of general transcription factors binding to TATA box

Answer 12

1. TFIID – comprise TBP + 10xTAFs - Binds/recognise TATA box. Recruits TFIIB
2. TFIIB provides a binding surface for Pol II and is involved in start-site determination
3. TFIIF is required for accurate initiation and recruits TFIIE and TFIIH – complex can start transcription
4. TFIIH binds and catalyses ATP-dependent unwinding of the start site DNA and phosphorylation go CTD of Pol II

Question 13

Metallothionein gene

Answer 13

Gene expressed in response to heavy metals – toxic
• Promoter can have more than 1 stimuli – have 4 sites to respond to metals
• To remove metals – induce gene to remove. Detection of heavy metals –> activate MTF1 and bind to consensus DNA sequence

Question 14

Oestrogen hormone signalling steps

Answer 14

1. Oestrogen binds to free oestrogen receptor complex –> oestrogen responsive element in promoter
2. Recruit GTFs to TATA box –> recruit RNA polymerase/start of transcription

Question 15

What are transcriptional activators and what do they contain.

Answer 15

Activators help to attract RNA Pol II to promoter

• Contain DNA binding domain and an activation domain. Bind through interactions with DNA binding co-factors.

Question 16

What is a transcription mediator

Answer 16

Complex that transduces both +/- regulatory info to the core transcriptional machinery

• Required to mediate the response to activators
• Interact with Pol 2 via CTD - increase efficiency or rate of PIC assembly.

Question 17

Types of Transcriptional co-activators

Answer 17

1. Those closely associated with the basal transcription machinery
2. Those that modify chromatin structure – histone acetyltransferases (HAT), chromatin remodelling complexes

Question 18

Steps in coactivators modifying chromatin structure

Answer 18

1. TATA inaccessible – gene activator protein recognises sequence and binds
2. Recognition allows the recruitment of HAT or chromatin remodelling complex
   - HAT – acetylate chromatin = chromatin disassemble
   - Chromatin remodelling complex – use ATP/energy to disrupt chromosomes
3. Allow RNA Pol II to recognise and bind to TATA box and transcription is active.