MBLG2 Lectures 9 to 15 · Cell cycle mutants • cell division cycle = cdc mutants – stuck at particular stages of the cell cycle • small = wee mutants – speed up cell cycle

! of !3 60 Lectures 9 - 15 MBLG 2071 !

GATED TRANSPORT transport between cytoplasm and nucleus (bi-directional)!controlled by the nuclear pore complex!active transport for macro molecules e.g. nucleic acids & proteins !active - costs the cell energy for transport!nuclear envelope and nuclear pore complexes for them barrier and the gate!nuclear import receptors are the adaptors between the gate and the cargo!signalling sequences (nuclear localisation signals, NLS) identify the cargo!!

The Nuclear Envelope!- continuous double membrane (inner + outer)!- the outer membrane extends into the

endoplasmic reticulum!!Nuclear Pore Complexes (NPCs)!- perforate the nuclear envelope!- there is an average of 200 NPCs in mammalians!- made up of approx. 30 proteins!- aqueous pore that doesn't require protein unfolding (like

transmembrane transport does)!- “tangles” block the central opening for large molecules (anything

above 40 kDa can’t get through in a significant number)!- free diffusion of small (<500 Da) molecules!- limited diffusion of medium sized (<60 kDa) molecules!!Nuclear Localisation Signal (NLS)!- typically a signal patch (hence transport of folded proteins)!- NLS contain positively charged amino acids, lysine and arginine!

� !nb: tumour suppressor p53 is often mutated in malignant tumour cases!!Nuclear Import Receptors!- large family of proteins whose job it is to bind directly to a protein that needs to go into the

nucleus, or via an adaptor protein which will then bind to transporting protein!- i.e. several receptors that bind either NPC and NLS of cargo protein (A), or NPC and a nuclear

import adaptor protein (B) [see image below]!

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Transport between cytosol and nucleus Gated transport

modified from: http://jcb-biowrites.rupress.org/2010/08/cell-biology-this-week-1.html

Nuclear pore complex (NPC) •  Free diffusion of small (<500

Da) and limited diffusion of medium-sized (<60 kDa) molecules

•  Regulates bidirectional active transport of large molecules (e.g. mRNA, proteins)

nucleus

cytoplasm


Nuclear Localisation Signal (NLS) •  Typically a signal patch (hence transport of folded proteins!)

•  NLS contain positively charged amino acids lysine and arginine

C

N

Bipartite signal patch

SV40 Large T antigen Pro-Lys-Lys-Lys-Arg-Lys-Val

Histone H2B Gly-Lys-Lys-Arg-Ser-Lys-Val Tumour suppressor p53 Lys-Arg-(aa)12-Lys-Lys-Lys

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The nuclear envelope

•  Continuous double membrane

•  The outer membrane extends into the ER

•  Nuc lear pore complexes (NPCs) perforate the nuclear envelope.

Fig 12-8



Nuclear pore complex (NPC) •  Made up of approx. 30 proteins

•  Aqueous pore that does not require protein unfolding (unlike transmembrane transport)

•  ‘Tangles’ block the central opening for large molecules

nucleus

cytoplasm

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The nuclear envelope

•  Continuous double membrane

•  The outer membrane extends into the ER

•  Nuc lear pore complexes (NPCs) perforate the nuclear envelope.

Fig 12-8



Nuclear pore complex (NPC) •  Made up of approx. 30 proteins

•  Aqueous pore that does not require protein unfolding (unlike transmembrane transport)

•  ‘Tangles’ block the central opening for large molecules

nucleus

cytoplasm

! of !6 60 Lectures 9 - 15 MBLG 2071

!!!!!!!!!!!!

Co-Translational Translocation!• Most proteins that are secretory, membrane-bound, or reside in the endoplasmic reticulum (ER),

golgi or endosomes use the co-translational translocation pathway!• This process begins with the N-terminal signal peptide of the protein being recognized by a

signal recognition particle (SRP) while the protein is still being synthesized on the ribosome!• The synthesis pauses while the ribosome-protein complex is transferred to an SRP receptor on

the ER (in eukaryotes) !• There, the nascent protein is inserted into the translocon, a membrane-bound protein conducting

channel (composed of the Sec61 translocation complex in eukaryotes) !• In secretory proteins and type I transmembrane proteins, the signal sequence is immediately

cleaved from the nascent polypeptide once it has been translocated into the membrane of the ER (eukaryotes) by signal peptidase. !

• The signal sequence of type II membrane proteins and some polytopic membrane proteins are not cleaved off and therefore are referred to as signal anchor sequences. !

• Within the ER, the protein is first covered by a chaperone protein to protect it from the high concentration of other proteins in the ER, giving it time to fold correctly. !

• Once folded, the protein is modified as needed (for example, by glycosylation), then transported to the Golgi for further processing and goes to its target organelles or is retained in the ER by various ER retention mechanisms.!!!

Post Translational Translocation!Three distinct scenarios for PTT into the ER:!a) soluble proteins!b) proteins with a single pass transmembrane domain (i.e. can only cross membrane once)!c) proteins with multi pass transmembrane domains (i.e. can cross several times)!!

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Co-translational translocation of proteins to the ER

Transport to the Endoplasmic reticulum Transmembrane transport

Fig 12-40

SRP = signal recognition particle

Three distinct scenarios for post-translational translocation to the ER:

(A) Soluble proteins

(B) Proteins with a single-pass transmembrane domain

(C) Proteins with multi-pass transmembrane domains

Transport to the Endoplasmic reticulum Transmembrane transport

! of !16 60 Lectures 9 - 15 MBLG 2071

� !* highly processive (high ability to remain associated with template strand)!* DNA pol ε synthesises the leading strand!* DNA pol δ synthesises the lagging strand!!

nb: there are many more DNA polymerases in eukaryotic replication however we will just focus on these!!

� !!!eukaryotes face the problem of how to replicate the ends of linear chromosomes = END REPLICATION PROBLEM!this is not a problem for prokaryotes as they have circular chromosomes!see picture: “incompletely replicated DNA” - this is where the last RNA primer was and the last Okazaki fragment. There is no way you can fill in this gap with normal DNA pol machinery, but if this gap is left, and replication occurs again (in daughter chromosomes), then the chromosome would just get shorter, and shorter again for the next generation, etc!!!

Telomeres Telomeres solve end replication problem!!recall that the ends of eukaryotic chromosomes are composed of repeated structures called Telomeres!in humans, the repeating sequence is 5’-TTAGGG-3’ (no need to remember this)!human telomeres can extend for as much as 10 kb!a telomere is double stranded for most of it, but there is a short single stranded section where 3’ end extends beyond 5’ end!!!!!

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Eukaryotic polymerases

•  DNA pol α/ primase – composed of two-subunit DNA pol α and two-

subunit primase –  low processivity (ability to remain associated

with template strand) –  lays down primer and initiates synthesis

•  DNA pol δ and ε – highly processive – elongation

Finishing replication

•  eukaryotes face the problem of how to replicate the ends of linear chromosomes = end replication problem

Figure 9.36 Watson et al. “Molecular Biology of the Gene” Seventh Edition

Finishing Eukaryotic Replication

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Eukaryotic polymerases

•  DNA pol α/ primase – composed of two-subunit DNA pol α and two-

subunit primase –  low processivity (ability to remain associated

with template strand) –  lays down primer and initiates synthesis

•  DNA pol δ and ε – highly processive – elongation

Finishing replication

•  eukaryotes face the problem of how to replicate the ends of linear chromosomes = end replication problem


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Telomeres •  recall from lecture 9 that the ends of eukaryotic

chromosomes are composed of repeating structures called telomeres

•  in humans the repeating sequence is 5′-TTAGGG-3′

•  human telomeres can extend for as much as 10kb

•  double stranded for most but short single stranded section where 3′ end extends beyond 5′ end


Telomerase •  recognises the overhang •  is a ribonucleoprotein – has both protein

and RNA components – protein: reverse transcriptase (kind of DNA

polymerase that uses RNA template to direct synthesis of DNA)

– RNA: includes ~1½ copies of the complement of the telomere sequence ie 5′-AAUCCCAAUC-3′

•  RNA component serves as a template for the reverse transcriptase component

! of !22 60 Lectures 9 - 15 MBLG 2071

!complex regulatory systems are in place to ensure orderly progression of the cell cycle!A novel price in physiology of medicine was awarded in 2001 to the discoverers of these regulatory systems (Leland Hartwell, Paul Nurse, Tim Hunt)!These prize winners used genetic and biochemical methods to identify the two classes of proteins that are key players in controlling the cell cycle!!

Yeast as a Model Organism ✤ Hartwell worked on saccharomyces cerevisae!✤ daughter cell grows as bud from mother cell (i.e. the bud will be the daughter cell coming off the

parent cell)!✤ followed progress of cell by observing bus growth !★Nurse worked on schizosaccharomyces bombe!★ grows longitudinally before dividing!★ followed progress of cell cycle by observing length of the cell !!Defining Mechanisms of Cell Cycle Control • identify genes that regulate progression through cell cycle!• but if such a gene is mutated, the cell cycle will be blocked and this will most likely be lethal

(and a dead yeast cell will not be helpful in the lab)!• circumvent this problem using conditional control!• conditional mutants display mutant phenotype under one growth condition but are normal

when grown under another condition!!� !Look for yeast that show cell cycle arrest (i.e. stop copying DNA and dividing) at high temperature (36 oC) but are normal at 23 oC!!!!!!

� !• cell division cycle mutants = CDC mutants!• they are yeast cells that are stuck as particular stages of the cell cycle!• wee mutants on the other hand, speed up cell cycle!• cdc and wee genes later found to encode kinases and phosphatases that play critical roles in

regulating specific stages of cell cycle !!

Temperature Sensitive Mutants

Cell Cycle Mutants - CDCs; Cdks

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Temperature sensitive mutants Look for yeast that show cell cycle arrest (ie

stop copying DNA and dividing) at high temperature (36 °C) but are normal at 23 °C

RIP

23 °C

permissive temperature

36 °C

restrictive temperature

Cell cycle mutants

•  cell division cycle = cdc mutants – stuck at particular

stages of the cell cycle •  small = wee mutants

– speed up cell cycle

cdc and wee genes later found to encode kinases and phosphatases that play critical roles in regulating specific stages of cell cycle

Figure 13.11 Molecular Cell Biology 4th Deition. Lodish et al., 2000

! of !23 60 Lectures 9 - 15 MBLG 2071

cdc2 - an example of a CDC mutant!- elongated cells at restrictive temperature!- cells have copied DNA!- these cells get to the end of G2 phase, but for some reason cannot get into M phase!- they were later found to be essential cells for transition into M phase from G2 phase!- cdc2 kinase (Cdk) is essential for transition from G2 phase to M phase!- hence, when the gene encoding this protein is mutated, cells arrest in G2 phase!- Cdk levels remain fairly constant throughout the cell cycle and are only activated through

interaction with the appropriate cyclin at specific stages!- groups of Cdks are names according to phase of the cell cycle in which they are active; G1/S

Cdk, S-phase Cdk, M-phase Cdk!- also have specific names e.g. cdc2!!

Cyclin dependent kinases play critical roles in regulating progression through the cell cycle (Hartwell and Nurse)

� !Tim Hunt worked with sea urchins and discovered a group of proteins whose level varied cyclically during the cell cycle!Hunt names these, cyclins!Groups of cyclins are names according to the phase of the cell cycle in which their levels are highest; G1/S cyclins, S-phase cyclins, M-phase cyclins!cyclins also have specific names e.g. cyclin B!!

Cyclins regulate the activity of the cyclin dependent kinases (Hunt) !

� !!Progression through each of the phases of the cell cycle is controlled by pairs of cyclins and

cyclin dependent kinases (Cdk)

Cyclins

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cyclins •  Tim Hunt worked

with sea urchins •  discovered a group

of proteins – level varied cyclically during cell cycle

•  called these cyclins

cyclins regulate the activity of the cyclin dependent kinases

www.nobelprize.org

http://en.wikipedia.org/wiki/Cyclin_E

cyclins and Cdks

Cdk

inactive

Cdk

active

cyclin

progression through each of the phases of the cell cycle is controlled by pairs of cyclins and cyclin dependent kinases (Cdk)

activated Cdk phosphorylates other proteins

Documents

MBLG2 Lectures 9 to 15 · Cell cycle mutants • cell division cycle = cdc mutants – stuck at particular stages of the cell cycle • small = wee mutants – speed up cell cycle