C7003 Cell and Molecular Biology Notes

Introduction to Cell and Molecular Biology

  • Dr. Greig Joilin, Assistant Professor in Biochemistry and Biomedicine, School of Life Sciences, University of Sussex.
  • Date: 27 January 2025

Learning Objectives

  • Summarize basic aspects of nucleic acid structure, gene expression, and gene expression regulation.
  • Summarize basic principles of protein structure and function.
  • Demonstrate knowledge of organelle functions in cells, contributing biological processes, and regulation of basic cellular processes.
  • Explain the basis of selected lab methods used to study cells/biomolecules; demonstrate ability to critically analyze experimental data.

Lectures

  • Lectures are twice on Mondays throughout the term, with two lectures on Tuesdays.
  • One lecture is online, the other in-person.
  • Attendance is recorded for engagement monitoring.
  • Topics are split into four areas:
    • Cell Cycle and DNA (Dr. Greig Joilin).
    • Transcription and Translation (Dr. Jon Baxter).
    • Protein Structure and Function (Prof. Louise Serpell).
    • Cell Organelles and Transport (Dr. Lorraine Smith).

Workshops

  • Two in-person workshops focused on techniques and their use in answering questions in cell and molecular biology.
  • Workshop 1: Weeks 4/5.
  • Workshop 2: Weeks 8/9.
  • Check Sussex Direct for stream assignments.
  • Attendance will be taken.

Practicals

  • Two practical sessions designed to provide experience with molecular biology lab work and develop data analysis skills.
  • Practical 1: Weeks 2-4, with a post-lab session the following Fridays.
    • Timetable clashes for some post-lab sessions will be addressed.
  • Practical 2: Weeks 6-7.
  • Session assignments may vary between the two practical sessions; check Sussex Direct.
  • Attendance will be taken.

Coursework

  • Due Weeks 7 and 10.
  • Two problem-based worksheets based on the two practical sessions.
  • Answer a set of questions based on generated data.
  • Each worksheet is worth 15% of the final mark.

MCQ Exam

  • Takes place during A2.
  • Consists of 40 questions covering lectures, practicals, and workshops.
  • Worth 70% of the final mark.
  • Guidance for coursework provided in practical sessions; MCQ covered in the revision session.

Module Communication

  • Important announcements via Canvas.
  • Content and assessment questions should be submitted to the Discussion page on Canvas.
  • Email queries limited to personal or confidential matters.
  • Responses generally provided within two working days.

Lecture 1: The Nucleus and the Nuclear Envelope

Background Reading

  • Essential Cell Biology available through PubMed NCBI Bookshelf.

Learning Outcomes

  • Understand that eukaryotic cells have a nucleus that compartmentalizes DNA away from the cytoplasm.
  • Know the composition of the nuclear membrane.
  • Understand theories on the origin of the nucleus.
  • Know that nuclear pore complexes allow the passage of macromolecules through the nuclear membrane, and how passage is regulated.
  • Understand that the nucleolus is a large sub-compartment of the nucleus where rRNAs are transcribed and ribosome subunits are assembled.

Synopsis

  • Eukaryotic cells have a nucleus that compartmentalizes DNA away from the cytoplasm, increasing process control but creating transport issues for large molecules (mRNA and proteins).
  • The nuclear pore enables molecule movement in and out.
  • This lecture discusses how this is achieved; uses ribosome biogenesis as an example.

Classifying Microorganisms

  • Carolus Linnaeus (1707-1778): Classified organisms into plant and animal kingdoms; revised since then.
  • Key categories: Fungi, Protozoa, Algae, Bacteria, Archaea, Viruses.
  • Prokaryotes:
    • Lack a nucleus and membrane-bound organelles.
    • Small, approximately 1.0 \, \mu m in diameter.
    • Simple structure.
    • Unicellular or acellular (viruses).
  • Eukaryotes:
    • Have membrane-bound organelles and a nucleus.
    • 10-100 \, \mu m in size.
    • Can be unicellular or multicellular.

Eukaryotic vs Prokaryotic cell size

  • Prokaryotes ~ 1 \, \mu m in diameter.
  • Eukaryotes ~ 10-100 \, \mu m in diameter.
  • Viruses ~ 10-100 \, nM.
  • Compartmentalization in eukaryotic cells allows them to become much larger and more complex than prokaryotic cells by focusing activities in distinct internal membrane-contained compartments.
  • Lecture focuses on the nucleus, the compartment where DNA is stored and processed.

The Eukaryotic Nucleus

  • In eukaryotic cells, the DNA is enclosed by two concentric membranes—a double membrane—that forms the nuclear envelope.
  • Visualization Techniques:
    • Immunofluorescence (I.F.) microscopy: Green indicates microtubules, blue indicates DNA (DAPI stain).
    • Electron microscopy: Provides a detailed picture of a slice through a eukaryotic cell.

Nuclear Membrane Structure

  • The outer nuclear membrane is contiguous with the endoplasmic reticulum (ER), connecting the space between the inner and outer nuclear membranes directly with the ER lumen.
  • The outer nuclear membrane is functionally similar to ER membranes but has slightly different protein composition.

Inner Nuclear Membrane and Nuclear Lamina

  • The inner nuclear membrane contains nucleus-specific proteins, including membrane proteins that organize the nuclear lamina.
  • The nuclear lamina is a fibrous network that provides structural support to the nucleus; without it, the nuclear membrane would fragment.

Evolution of Nuclear Membranes

  • The nuclear envelope and endoplasmic reticulum may have evolved through invagination of the plasma membrane.
  • The outer nuclear membrane’s continuity with the ER offers insights into its evolution.

Compartmentalization

  • Nucleus:
    • Transcription occurs: \text{DNA} \rightarrow \text{RNA}
  • Cytoplasm:
    • Translation occurs: \text{RNA} \rightarrow \text{protein}
  • Transport across nuclear membranes is essential because:
    • mRNA must be exported from the nucleus to be translated into protein.
    • Transcription and replication require enzymes and proteins that must be imported into the nucleus.

Nuclear Pore Complexes

  • The nuclear membrane is studded with nuclear pore complexes, which are the only channels through which polar molecules and macromolecules can pass through the nuclear envelope.

Structure of Nuclear Pores

  • Nuclear pores are large multi-protein complexes composed of about 30 different proteins.
  • Negatively stained electron microscopy reveals a structure with eight-fold symmetry around a large central channel.
  • Protein fibrils protrude from both sides, forming a basket-like structure on the nuclear side.
  • Many nuclear pore proteins contain unstructured regions that form a jumbled meshwork, filling the channel center.
  • Proteins over 5 kD are blocked from passively diffusing through.

Nuclear Localization Signals (NLS)

  • A nuclear localization signal (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 and 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.
  • Ran-GTP covers a loop (red) important for NLS binding.

Cycling of Ran Across the Nuclear Envelope

  • Ran-GAP in cytosol.
  • Ran-GEF in nucleus.

Nuclear Export

  • Nuclear export signal recognized by exportin (similar to importins).
  • Ran-GTPase binds to exportins with cargo to promote export through the pore.
  • GTP/GDP switch occurs upon entering the cytosol.
  • RAN-GAP is cytoplasmic.
  • RAN-GEF is nuclear.

Nuclear Import and Export Dynamics

  • Localization depends on the balance between import and export processes.
  • Example: NFAT-GFP (Nuclear factor of activated T-cells).

Nucleolus

  • NFAT- GFP (Nuclear factor of activated T-cells)
  • The most prominent structure in the nucleus is the electron-dense nucleolus
  • It is the 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’s Large subunit is composed of both RNA and protein.

Ribosome 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.

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

rRNA genes 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
  • Compartmentalisation
    • 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 recognise the localisation 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 Reminders

  • 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 to us will also be added here to prevent us having to repeat answers
  • 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. Questions