Cell and Molecular Biology Flashcards

Intro to C7003: Cell and Molecular Biology

• Dr. Greig Joilin, Assistant Professor in Biochemistry and Biomedicine, School of Life Sciences, University of Sussex

• Cell and Molecular Biology, 27 January 2025

Learning Objectives for the Module

• Summarize basic aspects of nucleic acid structure, how genes are expressed, and basic principles of how gene expression is regulated.

• Summarize the basic principles of protein structure and function.

• Demonstrate knowledge of the functions of organelles in cells and the biological processes to which they contribute, and an understanding of the regulation of basic cellular processes.

• Explain the basis of selected laboratory methods used to study cells and biomolecules and demonstrate an ability to process and critically analyze experimental data.

MCQ Exam

• This will take place during A2.

• It will consist of 40 questions covering the lectures, practicals, and workshops.

• Worth 70% of your final mark.

• Guidance for the coursework will be provided in the practical sessions, and the MCQ will be covered in the revision session at the end of the module.

LECTURE 1: The Nucleus and the Nuclear Envelope

• Reading: Biological Sciences pp 384 to 388.

• Essential Cell Biology – Molecular Biology of the Cell - https://www.ncbi.nlm.nih.gov/books/NBK26932/

• Dr. Jon Baxter

• Background reading from Essential Cell Biology - Molecular Biology of the Cell available through PubMed Bookshelf https://www.ncbi.nlm.nih.gov/books

Learning Outcomes

• By the end of this lecture you will have covered:

  • That eukaryotic cells have a nucleus that compartmentalizes the DNA away from the cytoplasm.

  • The composition of the nuclear membrane

  • Theories on the origin of the nucleus

  • That nuclear pore complexes allow passage of macromolecules through the nuclear membrane and how passage is regulated.

  • That the nucleolus is a large sub-compartment of the nucleus where rRNA’s are transcribed and ribosomes subunits assembled.

Synopsis

• Eukaryotic cells have a nucleus that compartmentalizes the DNA away from the cytoplasm. This increases control of processes but creates a problem as large molecules (mRNA and proteins) have to move in and out of the nucleus.

• The nuclear pore enables movement of molecules in and out. This lecture will discuss how this is achieved and use the example of ribosome biogenesis to illustrate the process.

Classifying Microorganisms

• Carolus Linnaeus (1707-1778): Classified organisms into plant and animal kingdom.

  • Key categories: Fungi, Protozoa, Algae, Bacteria, Archaea, Viruses

• Prokaryotes: Lack nucleus and membrane-bound organelles, are small ~$1.0 \mu m, with a simple structure, unicellular or acellular (viruses)

• Eukaryotes: Have membrane-bound organelles and nucleus, ~$10-100 \mu m, can be uni or multicellular

• The compartmentalization of the eukaryotic cell allows the cells to become much larger and more complex than prokaryotic cells, in part through focusing different activities in distinct internal membrane contained compartments. Focus on the compartment where the DNA is stored and processed – the nucleus.

The Eukaryotic Nucleus

• In eukaryotic cells, the DNA is enclosed by two concentric membranes – a double membrane – that forms the nuclear envelope

• Immunofluorescence (I.F.) microcopy of the cell.

  • Green – microtubules

  • Blue – a DNA stain ((DAPI))

• Electron microscopy picture of a slice through a eukaryotic cell

The Outer Nuclear Membrane

• The outer nuclear membrane is contiguous with the endoplasmic reticulum (ER), so the space between the inner and outer nuclear membranes is directly connected with the lumen of the ER.

• The outer nuclear membrane is functionally similar to the ER membranes, but differs slightly in protein composition.

The Inner Nuclear Membrane and the Nuclear Lamina

• The inner nuclear membrane carries nuclear specific proteins such as the membrane proteins that organize the nuclear lamina, a fibrous network that provides structural support to the nucleus.

• Without this network the nuclear membrane will fragment.

Evolution of Nuclear Membranes

• The nuclear envelope and endoplasmic reticulum may have evolved through invagination of the plasma membrane.

• The outer nuclear membrane is continuous with the endoplasmic reticulum (ER) which gives us a pointer as to how it evolved.

Compartmentalization

• Nucleus: transcription (DNA → RNA)

• Cytoplasm: translation (RNA → protein)

• Transport across nuclear membranes is essential.

  • mRNA has to be exported from the nucleus to be translated into protein

  • Transcription and replication require enzymes and these proteins have to be imported into the nucleus

The Nucleus and Cytoplasm

• The nuclear membrane is studded with nuclear pore complexes that are the sole channels through which polar molecules and macromolecules pass through the nuclear envelope

Nuclear Pores

• Nuclear pores are large multi-protein complexes composed of about 30 different proteins

• Negatively stained electron microscopy of purified nuclear pores reveals a structure with eight-fold symmetry organized around a large central channel

• Protein fibrils protrude from both sides of the complex, and on the nuclear side they form a basket-like structure.

• Many nuclear pore proteins contain extensive unstructured regions that form a jumbled meshwork that fills the center of the channel

• Proteins over 5kD are blocked from passively diffusing through.

Nuclear Localization Signals

• A nuclear localization signals (NLS) is a protein tag that identifies proteins destined for the nucleus

• The tag typically consists of one or two short sequences containing positively charged Lysine residues

Nuclear Transport Receptors

• Importins carry proteins into the nucleus

• Exportins carry proteins out of the nucleus

Energy for Nuclear Transport

• The energy supplied by GTP hydrolysis drives nuclear transport

Ran-GTP Binding

• Ran-GTP binding leads to cargo release

• Nuclear transport receptors are made up of repeated α-helices that stack into large arches or snail-shaped coils

• Cargo and Ran-GTP bind different arches

• RanGTP covers loop (red) which is important for NLS binding

Cycling of Ran

• Cycling of Ran across the nuclear envelope

Export

• Nuclear export signal

• Recognized by Exportin (similar to importins)

• In this case RAN-GTPase binds to exportins with cargo to promote export through pore

• GTP/GDP switch – on entering cytosol

• RAN-GAP cytoplasmic

• RAN-GEF nuclear

Nuclear Import and Export

• Nuclear import and export is dynamic

• Localization depends on balance between the two processes

  • NFAT- GFP (Nuclear factor of activated T-cells)

Internal Organization of the Nucleus – The Nucleolus

• The most prominent structure in the nucleus is the electron dense nucleolus

• Site of ribosome biogenesis where transcribed and processed rRNA are combined with proteins to form the ribosomal subunits

Ribosomes

• Ribosomes translate mRNA into proteins in the cytoplasm (rough ER and in cytosol).

• The ribosome is a very large and complex structure, composed of two-thirds RNA and one-third protein.

Ribosome Subunits

• 2 subunits:

  • Small subunit: Platform where tRNAs are matched to the codons of the mRNA

  • Large subunit: Catalyzes the formation of the peptide bonds linking amino acids into a polypeptide chain

  • Sub-units come together on an mRNA for translation

• The cells require a lot of ribosomes. Therefore, the rRNA genes are highly transcribed.

The Nucleolus and rRNA Genes

• The nucleolus is organized around chromosomal regions containing the rRNA genes

• In higher eukaryotes, the ribosome contains four types of ribosomal RNAs (rRNAs), 5S, 5.8S, 18S, and 28S

• The genes encoding the 5.8S, 18S, and 28S rRNAs are clustered in the genome in large tandem arrays

rRNA Genes

• rRNA genes are in tandem arrays

• This classic image shows the transcription of the repeats of the rRNA cluster with each transcription unit (TU) in the tandem array separated by a non-transcribed spacer (NTS)

Processing of rRNAs

• The 5.8S, 18S and 28S rRNAs are transcribed as a single 45S pre-rRNA that is then sequentially cleaved to give rise to the three mature rRNAs

• The 5S rRNA is encoded by a separate gene

• rRNA processing occurs in the nucleolus

Ribosome Assembly and Export

• Ribosomes are assembled in the nucleus and exported to the cytoplasm

• Ribosomes are composed both of rRNAs and proteins that interact with the rRNAs.

• Ribosomal protein genes are transcribed in the nucleus, and translated in the cytoplasm

• The proteins are imported into the nucleus and assemble on the pre-rRNA in the nucleolus

• As the rRNAs mature, additional ribosomal proteins are added to form pre-ribosomal particles

• These particles are exported from the nucleus via the nuclear pores to yield active ribosomal subunits

Key points: Nuclear Envelope

• The DNA in prokaryotic cells occupies the same compartment as the cytoplasm

• In eukaryotic cells, the DNA is contained in a membrane bound compartment – the nucleus

• The nuclear envelope consists of an outer and inner membrane

  • The outer membrane is contiguous with the ER and is similar in composition

  • The inner membrane is enriched in nucleus-specific proteins and organizes the nuclear lamina

• Compartmentalization

  • transcription (mRNA) in nucleus,

  • translation (protein) in cytoplasm,

Key points: Nuclear Pore

• Nuclear pore complexes regulate transport across the nuclear envelope

• Proteins targeted for the nucleus contain a nuclear localization signal

• Receptors called importins or exportins recognize the localization signals and transport proteins through the nuclear pore

• GTP hydrolysis by the Ran-GTPase drives transport in and out of the nucleus

Key points: Nucleolus

• The nucleolus is an aggregation of rRNA gene clusters

• It is the site of ribosome assembly

• Ribosomal proteins assemble on pre-rRNAs in the nucleolus

• pre-ribosome particles are then exported through the nuclear pores

Module Communication (Repeated)

• Important announcements will be conveyed through Canvas – please pay attention to these as they will contain important messaging for this module.

• Questions about the content and assessments should first be submitted to the Discussion page on Canvas.

  • Any questions emailed will also be added to the discussion page to prevent repetition.

• Please limit email queries to those of a personal or confidential nature.

• Responses to emails and discussion questions will be provided generally within two working days.

Chromosomes and Chromatin

• Reading: Biological Sciences pp 206 to 211

• Essential Cell Biology – Molecular Biology of the Cell

  • https://www.ncbi.nlm.nih.gov/books/NBK26834/

  • https://www.ncbi.nlm.nih.gov/books/NBK26847/

  • Fig 4.45 to end

Learning Outcomes

• By the end of this lecture you should be able to know:

  • The types of non-coding and coding sequences that make up eukaryotic chromosomes.

  • How eukaryotic chromosomes vary in their length, make up and organization in the nucleus

  • How chromosomes are organized by nucleosomes and how nucleosome fibre organization occurs at multiple levels

  • How epigenetic marks and association with different types of chromatin alter gene expression

Synopsis

• This lecture aims to explain how the genetic information is arranged on eukaryotic chromosomes, including discussion of non-coding DNA where the functions are either essential for genetic inheritance or where DNA has unclear or possibly no strong evolutionary function.

• Discuss how DNA is organized by histones into chromatin and how chromatin organization varies both during the cell cycle and also according to transcriptional activity

Organisation of the Genome

• Prokaryotic cells contain a single circular genome which is coiled up to allow it to fit within the intracellular space. Prokaryotic cells may also contain plasmids, small circular double stranded DNAs that replicate independently of the main genome.

• The linear double stranded chromosomes of eukaryotes are compartmentalized in the nucleus. The number and size of chromosomes in the nucleus vary between species. Eukaryotic cells cells contain homologous pairs of chromosome (each pair is a different colour). Outside the nucleus the mitochondria also contain multiple copies of a double stranded circular genome.

Eukaryotic vs Prokaryotic Genomes

• Eukaryotes genomes are larger and more complex than prokaryotic

• No clear correlation between genome size and complexity of the organism

Eukaryotic Chromosomes

• In eukaryotes, the genome is broken up into long, linear, double-stranded structures called chromosomes

• The human genome has 24 chromosomes

  • 22 autosomes and 2 sex chromosomes

• Human cells are diploid (2n = 46)

Chromosome Number

• Chromosomes are arbitrary units of heredity

• Even closely related species can have different numbers of chromosomes

• There is no simple relationship between gene number, chromosome number, and genome size

Repeated and Unique Sequences

• Eukaryotes genomes are often larger, more complex and more variable in size because they all have differing amounts of various non-coding DNA sequences including:

  • More genes which contain introns

  • More regulation e.g. cell type specific expression

  • More ‘junk’ DNA – Repeated sequences; transposons, simple repeat and duplications. Often associated with heterochromatin

• Also contain multiple functional DNA elements – telomeres, centromeres, origins of replication.

Functional Chromosome Elements

• Telomeres – stabilize ends

• Replication origins – where the duplication process of the DNA starts

• Centromere – to segregate the sister chromosome

Telomeres

• Telomeric repeat sequences are found at the ends of chromosomes.

• Provide a buffer between gene sequences and end of chromosome.

• Bound by proteins that help protect the ends from exposure.

Replicated Chromosome

• Multiple origins of replication, at least one per arm

• After replication there are two identical sister chromatids

• Each chromatid contains one old and one new strand (semi-conservative replication)

• Sisters are held together until chromosome segregation during mitosis

Centromeres

• Centromeres are normally associated with arrays of repeated DNA sequences.

• In humans the alpha satellite repeats (red) are AT-rich and vary slightly from one another in their DNA sequence.

• Blue is flanking centric heterochromatin, which contains DNA sequences composed of different types of repeats.

• Kinetochore positioned within inner repeats.

• The kinetochore consists of an inner and an outer plate, formed by a set of kinetochore proteins.

• The spindle microtubules attach to the kinetochore in M phase of the cell cycle

Chromosome Organization

• In Interphase nuclei, the chromosomes are distributed throughout the nucleoplasm

• At mitosis the chromosomes condense - individual chromosomes can be seen with the light microscope

Chromosomes in the Interphase Nucleus

• During interphase, individual chromosomes are not obviously structured in a stable conformation, but they are still partially resolved from one another across the nucleus (chromosome territories).

• This arrangement minimizes tangles between the chromosomes and helps condensation and segregation during mitosis

The Rabl Configuration

• In some cell types , (especially in plant cells) chromosomes adopt the Rabl configuration with the centromeres clustered at one end, and the telomeres abutting the nuclear envelope at the opposite pole reflecting the arrangement of the chromosomes at anaphase

Chromosomal DNA

• Packaging problem - 2m DNA into 6um human cell nucleus

Mitotic Chromosomes

• Mitotic chromosomes can be up to 10,000x more compact than the length of the DNA

• Therefore, how the DNA is packaged in the cell is highly variable.

The Nucleosome

• At the simplest level individual nucleosomes are arranged on the DNA like beads on a string.

• DNA isolated from an interphase nucleus also appears as a 30 nm thick fibre – likely more variable thickness in vivo

• DNA is compacted by association with proteins - chromatin

Nucleosome Structure

• The nucleosome core particle consists of 147 base pairs of DNA wound around a protein core

• The protein core is made of a small highly basic proteins called histones

• The octameric histone core contains two molecules of each of four histones, H2A, H2B, H3 and H4

Histone Structure

• H3 (green) and H4 (blue) tetramer on top and form a scaffold onto which two sets of H2A(red)-H2B(orange) dimers are added (bottom).

• 147bps of DNA is wrapped around the octamer in a sequence non-specific manner (though some sequences wrap better than others, so spacing between nucleosome varies slightly).

Histone H1

• Histone H1 binds to both the DNA and nucleosome in the are where the DNA exits the nucleosome stabilizing the chromatin fibre

Higher Order Packaging

• DNA is wound around histones

• An additional histone, Histone H1, arranges nucleosomes into the 30 nm fibre

• During mitosis, this 30 nm fibre is further condensed at two additional levels to achieve the packing that is observed in mitotic chromosomes

Packaging Variation

• During interphase the extent of packaging varies in different regions of the genome.

• By electron Microscopy, the chromatin in the nucleus appears in two forms:

  • Euchromatin: a diffuse open configuration, and

  • Heterochromatin: a condensed electron dense form.

• Euchromatin is transcriptionally active – relatively loose nucleosome arrangement

• Heterochromatin is transcriptionally inactive associated chromatin conformations that are highly compacted – inactive genes, long repeat sequence arrays or regions adjacent to repeats.

Interphase Chromatin

• Interphase chromatin is very dynamic

• Decondensed or open chromatin (= euchromatin) is associated with transcriptionally active regions of the genome

• Closed Condensed chromatin (= heterochromatin) is associated with transcriptionally repressed regions of the genome

Histone Remodelers

• Histone remodelers promote sliding of histone DNA to promote either euchromatin or heterochromatin

Chromatin Structure Along Chromosome

• Chromatin condensation can vary along the length of the chromosome

• Telomeres and centromeres are typically heterochromatic. These regions are also gene poor.

• The gene-rich regions of the chromosomes are typically euchromatic.

• Regional modifications produce blocks of different degrees of compaction

Histone Modification

• Each core histone has a ‘tail’ that extends out of the nucleosome core.

• These tails can be modified by methylation and acetylation to influence chromatin structure and thus gene expression

Gene Expression

• Histone modifications can change overall chromatin structure by either directly affecting how nucleosomes pack together or by promoting or inhibiting the binding of proteins that can alter chromatin organisation

Heterochromatin Spreading

• Heterochromatin spreading requires reader–writer coupling – one protein recognizes and binds to the modified histone.

• The bound protein then recruits a histone modifying enzyme which modifies neighboring histones

Epigenetic Marks

• Heterochromatic chromatin modifications spread from the telomere into telomeric adjacent gene frequently heritably silencing them.

Heterochromatin Establishment

• DNA binding proteins (DBPs) can recruit histone modifying enzymes (HME) to chromatin

• A nascent transcript generated by RNA polymerase can contain recognition motifs for either ribonucleoprotein (RNP) or RNA binding proteins (RBP) which recruit histone modifying enzymes (HME) to chromatin

X Chromosome Inactivation

• To ensure equal gene expression between XY and XX individuals the extra X is transcriptionally silenced in early development.

• The silenced, heterochromatic state is then inherited throughout the rest of development.

• In mammals establishment occurs by expression of the non coding RNA Xist on the X chromosome that will be inactivated.

Implications of X Chromosome

• When random X inactivation occurs in the early embryos different X chromosomes are inactivated in the embryonic cells.

• If different X chromosomes have slightly different genes these differences are maintained in the clonal offspring of the embryonic cell

• For example, in cats, a fur pigmentation gene is X-linked. In Calico cats, different versions of the gene controlling fur colour are on the X chromosomes. Depending on which copy of the X chromosome each cell chooses to leave active, either an orange or black coat color results. These differences are only observed in animals with multiple X chromosomes, which explains why almost all Calico cats are female

Organization Matters

• Prokaryotes genomes small, few genes, single circular chromosome, one origin of replication

• Eukaryotes genomes are larger, more genes linear chromosomes, many origins of replication, telomeres and centromere

• Eukaryote nucleus:

  • The interphase nucleus is highly ordered with chromosomes occupying particular positions

  • DNA is highly condensed even during Interphase

  • This condensation is achieved by interaction with proteins

  • DNA + histones = chromatin

Chromatin Packaging

• The fundamental unit of chromatin is the nucleosome

  • DNA wrapped round histone core

• Packing of nucleosomes leads to higher orders of condensation

• Chromatin exists in two forms:

  • Euchromatin = ‘open’ configuration, gene rich, actively transcribed

  • Heterochromatin = electron-dense ‘closed’ configuration, inactive

• The open and closed configurations are regulated by modification of histone protein tails

• Chromatin is very dynamic, chromatin remodelers move nucleosomes to expose DNA

Module Communication (Repeated Again)

• Important announcements will be conveyed through Canvas – please pay attention to these as they will contain important messaging for this module.

• Questions about the content and assessments should first be submitted to the Discussion page on Canvas.

  • Any questions emailed will also be added to the discussion page to prevent repetition.

• Please limit email queries to those of a personal or confidential nature.

• Responses to emails and discussion questions will be provided generally within two working days.

Lecture: Mitosis and Meiosis

• Biological Sciences: Hard copy and ebook pp413-417 and pp416-424

• Molecular Biology of the Cell Online edition

  • https://www.ncbi.nlm.nih.gov/books/NBK26934/

  • https://www.ncbi.nlm.nih.gov/books/NBK26931/

  • https://www.ncbi.nlm.nih.gov/books/NBK26840/

Learning Outcomes

• By the end of this unit of teaching and learning, you will be able to identify and understand:

  • How interphase prepares cells for cell division during mitosis

  • The different visual stages of mitosis

  • The molecular processes that are driving the visual changes of mitosis

  • How cells initiate the metaphase to anaphase transition and end mitosis

  • The different stages of meiosis

  • How meiosis generates genetic diversity

  • How errors in meiosis can lead to developmental problems such as Down Syndrome

Multicellular Organism

• Most cells in a developed multicellular organism are in a G0 state.

• In multicellular organisms, during development, growth and wound healing, cells receive signals from their environment to enter into the cell cycle and divide.

• This sets off a chain of exquisitely coordinated events which culminate in the cell passing through mitosis when a full complement of chromosomes are duplicated and segregated into the the two halves of the cell just before it divides to form two cells

• If cell division is not carried out correctly and the daughter cells do not get a full complement of genetic information, the cells will die.

Interphase Table

Mitosis

• Mitosis though a microscope.

  • Red colour is DNA stained with propidium iodide

  • Green colour is microtubules marked with GFP

Mitosis and Cytokinesis

• Careful examination of mitotic cells allowed to be broken up into distinct stages;

Mitosis Stages

• Prophase

  • At prophase, the replicated chromosomes, each consisting of two closely associated sister chromatids, condense.

  • Outside the nucleus, the mitotic spindle assembles between the two centrosomes, which have begun to move apart.

• Prometaphase

  • Prometaphase starts abruptly with the breakdown of the nuclear envelope.

  • Chromosomes can now attach to spindle microtubules via their kinetochores and undergo active movement.

• Metaphase

  • At metaphase, the chromosomes are aligned at the equator of the spindle, midway between the spindle poles.

  • The paired kinetochore microtubules on each chromosome attach to opposite poles of the spindle.

• Anaphase

  • At anaphase, the sister chromatids synchronously separate, and each is pulled slowly toward the spindle pole it is attached to.

  • The kinetochore microtubules get shorter, and the spindle poles also move apart, both contributing to chromosome segregation.

• Telophase

  • During telophase, the two sets of chromosomes arrive at the poles of the spindle.

  • A new nuclear envelope reassembles around each set, completing the formation of two nuclei and marking the end of mitosis.

  • The division of the cytoplasm begins with the assembly of the contractile ring.

• Cytokinesis

  • During cytokinesis of an animal cell, the cytoplasm is divided in two by a contractile ring of actin and myosin filaments, which pinches in the cell to create two daughters, each with one nucleus.

Compact Chromosomes

• Condensin complexes (blue) bind to chromosomes at the beginning of mitosis and fold them into consecutive cis-loops until they become highly compacted and condensed (activity of Condensin is upregulated by mitotic CDK kinases).