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

• Reading:
  • Biological Sciences, pp. 384-388.
  • Essential Cell Biology - Molecular Biology of the Cell (available through PubMed).
    • https://www.ncbi.nlm.nih.gov/books/NBK26932/

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