Cell Biology: Active Transport and DNA Replication

Nutritional Biochemistry

• Course code: DIET413/BHCS1019
• Lecturer: Dr Nathaniel Clark FHEA RNutr MRSB
• Email: [email protected]

Questions

• Question 1: Proteins spanning the membrane, composed of alpha helices, contain specific amino acid side chains central to their function.
• Question 2: Transport proteins moving solutes against concentration gradients utilize carrier proteins rather than channel proteins. This is because carrier proteins undergo conformational changes and bind to the transported molecule.
• Question 3: Simple diffusion is the passive movement of molecules down their concentration gradient, while facilitated diffusion involves specific transport proteins (carrier or channel) aiding this movement.
• Question 4: Active transport is powered by chemical energy (ATP) and electrochemical gradients (like the Na+-K+ pump).
• Question 5: The similar structure of K+ and Na+ channels creates a common orientation in the cell membrane. However, the different ion selectivity within each channel allows specific ions to pass through. Different selectivity arises from differences in amino acids within the channel structure.

Learning Outcomes

• Outcome 1: Describe DNA structure and organisation in the nucleus.
• Outcome 2: Describe DNA replication.
• Outcome 3: Outline transcription and translation of a gene into a protein.
• Outcome 4: Briefly outline the structure, control, and role of mtDNA, and linked diseases.

Genes and Genetics

• Humans have 100 trillion cells, each nucleus (except red blood cells) has 46 chromosomes arranged in 23 pairs.
• Chromosomes contain genes, which are sections of DNA that code for proteins.
• Each chromosome comes from each parent.

The Genome

• Every cell in an organism contains essentially the same genetic information in the genome.
• The genome controls biochemical activities and is also responsible for inherited traits.
• The human genome was sequenced in 2003 and contains roughly 20,000-21,000 genes.
• The genomes of most humans are 99.9% identical but 0.1% accounts for variation and diseases.
• Individual genes control the synthesis of specific proteins.

The Structure of DNA

• DNA is a polymer of nucleotides.
• DNA is a double helix, analogous to a ladder or a twisted ladder shape.
• This helix has two nanometers in width and 10.5 steps for every three point four nanometers.
• DNA twists like an alpha helix.

DNA Methylation

• DNA methylation involves the addition of a methyl group to cytosine nucleotides.
• Demethylation is the removal of a methyl group.
• This process has profound effects on gene expression and is considered part of epigenetics.

Nucleosides

• Nucleosides consist of a sugar and a nitrogenous base.
• The sugar and base are linked together through a bond.
• Purines (Adenine and Guanine) have two rings. Pyrimidines (Cytosine and Thymine) have one ring.

Nucleotide Structure

• Nucleotides are the building blocks of DNA and consist of a sugar, a phosphate group, and a nitrogenous base—these bases form the "steps" of the double helix.
• DNA has deoxyribose sugar, while RNA has ribose.

Hydrogen Bonding Between Bases

• The specific pairing of bases (A with T, G with C) is crucial and is driven by hydrogen bonds.
• This base pairing is essential in DNA replication and transcription.

Polynucleotides

• Nucleotides link together through phosphodiester bonds to form polynucleotides, forming the backbone of DNA and RNA.
• This bonding creates a chain of nucleotides and can be described as a phosphate-sugar backbone.

The DNA Double Helix

• DNA is a double helix, where the two strands run in opposite directions, described as antiparallel.
• Deoxyribose are on the inside, carbons on one strand being above the carbon parts of another strand.
• The phosphate group is outside.

Histones, Chromatin and Chromosomes

• DNA is packaged into chromatin to fit inside the cell's nucleus, with histones helping this compaction.
• Histone proteins form nucleosome particles.
• Nucleosomes condense to form chromatin fibres.
• Chromatin fibres further condense to form chromosomes.

Structure of Genes

• Genes are discrete segments of DNA and encode the amino acid sequence of polypeptide chains.
• Telomeres cap chromosome ends to protect from damage and fusion.

Activity

• Students were asked to discuss the chemical differences at the ends of DNA sequences and how this affects DNA structure.
• Students were also asked to create lists of key words associated with DNA structure.

The Cell Cycle

• DNA replication is part of a larger cycle of cell events.
• The cycle includes phases like G1 (growth), S (synthesis), G2 (further growth), and M (mitosis).
• Precise DNA replication occurs in the S phase.
• Growth of the cell occurs in G2 and mitosis/cell division in M phase.

The Cell Cycle and Checkpoints

• The cell cycle has checkpoints to ensure accuracy of replication and growth.
• Three checkpoints are described: • Cell Growth Checkpoint (G1) • DNA Synthesis Checkpoint (S) • Mitosis Checkpoint (M)

DNA Replication

• DNA replication is the process of making a copy of DNA.
• Replication uses the existing strands as a template to create new, complementary strands.
• There is a leading strand that is synthesized in the 5' to 3' direction continuously and a lagging strand that is synthesized discontinuously in short sections called Okazaki fragments.

DNA Replication - Unzipping

• Helicase separates the two DNA strands.
• DNA polymerase requires a short stretch of RNA known as a primer to begin synthesis in the 5'—>3' direction.

DNA Replication - The Replication Fork

• The replication fork is the region where DNA unwinds.
• DNA synthesis occurs only in the 5'->3' direction.

DNA Replication - Leading/Lagging Strands

• Leading strand is synthesized continuously in the 5'—>3' direction away from the replication fork.
• Lagging strands are synthesized discontinuously away from the replication fork in Okazaki fragments.

DNA Replication - Lagging Strand

• The gap between Okazaki fragments is then closed by another enzyme, DNA ligase joining the fragments together using phosphodiester bonds.
• The resulting complementary strands of DNA are identical to the original DNA.

DNA Replication - Storage

• Once DNA replicates, it must reorganize to be suitable for the two daughter cells.
• The reorganization makes different structures depending on the cell cycle.
• This reorganisation includes the formation of chromosomes.

From Genes to Proteins

• DNA molecules contain genes, the blueprint for proteins.
• Each gene is a small section of a DNA molecule which directs the synthesis of specific proteins.
• The process of protein synthesis is normally regulated by hormones and growth factors.

Transcription

• The process of copying information from DNA into mRNA for protein synthesis is Transcription.
• This process happens with special sequence that makes the polymerase enzyme locate the desired site, then the enzyme binds and starts transcribing.

Initiation of Transcription – TFs

• General transcription factors (TFs) are proteins that bind at the start of genes to initiate transcription.
• In mammalian cells, these proteins control the rate of transcription.
• TFs typically bind to regulatory sequences in the DNA known as promoters and enhancers, which control the transcription process.

Initiation of Transcription – Other Regions

• Enhancers are DNA elements located further away from the start of the gene.
• They work through binding to protein factors that influence the rate of transcription.
• The TATA box is a sequence near the start of most genes, which helps to position the enzyme for initiation.

Chain Initiation

• During initiation, RNA polymerase binds to a specific region of DNA called the promoter, and initiates the addition of nucleotides to form the initial RNA molecule.

Chain Elongation

• RNA polymerase moves along the coding DNA strand, adding complementary nucleotides in a 5' to 3' direction.
• Only one strand of DNA is used as a template for RNA synthesis.

Chain Termination

• Termination of RNA synthesis occurs because of special sequences in DNA that signal RNA polymerase to detach from the DNA and release the RNA strand.

Post-Transcriptional Modification

• The initial RNA transcript, called pre-mRNA, undergoes modifications before becoming mature mRNA.
• Introns, non-coding regions of DNA are removed.
• Exons, coding regions of DNA, are joined.

Translation

• The process of assembling amino acids into a protein from the information encoded in mRNA is Translation.
• Ribosomes are the complex structures that support this process in the cell.
• Ribosomes bind to mRNA to translate the nucleotide sequence into the amino acid sequence of a protein.

Ribosomes

• Ribosomes are the cellular structures responsible for protein synthesis.
• Ribosomes are composed of ribosomal RNA (rRNA) and proteins.
• There are binding sites on ribosomes, such as amino acid binding sites and three other sites involved in tRNA interactions with mRNA. This allows for the binding of amino acids corresponding to the specific mRNA code.

Transfer RNA

• The tRNA molecules bring amino acids to the ribosome, according to the mRNA codons.
• Each tRNA has a specific anticodon that binds to a corresponding mRNA codon.
• The A and P sites are essential binding sites on the ribosome where tRNA molecules (carrying amino acids) engage during protein synthesis.

The Triplet Code

• A codon is a three base sequence (bases = A, U, G, C) that constitutes a message to be transferred or acted on for protein synthesis.
• The codons dictate the sequence of amino acids in proteins which make up cells.

Elongation Peptide Chain

• The ribosome moves along the mRNA, adding amino acids one by one to the growing polypeptide chain.
• The following amino acid is delivered to the A site of the ribosome, where it is added to the growing polypeptide chain.

Termination

• Translation ends when a stop codon is encountered on the mRNA.
• Release factors bind to the stop codon, causing the ribosome to dissociate and release the completed polypeptide chain.

Mitochondrial DNA (mtDNA)

• Mitochondria have their own DNA and are important in cellular energy production (ATP).
• mtDNA encodes for 13 proteins for oxidative phosphorylation as well as rRNAs and tRNAs.
• mtDNA is a circular double-stranded DNA molecule that is different in structure to nuclear DNA.

Mitochondrial DNA Replication

• mtDNA replication is similar to nuclear DNA replication though some key differences exist.
• The timing of lagging strand production is a debatable topic. There are two theories; one supports strand-asymmetric replication, whereas another supports strand-coupled replication.

Transcription and Replication of mtDNA

• mtDNA transcription initiation requires specific proteins such as RNA polymerase and a twinkle helicase.
• mtDNA replicating and transcribing machinery is similar, at a fundamental level, to processes in the nucleus.

Transcription and Replication - Differences

• Mitochondrial DNA lacks introns and non-coding sequences between genes.
• mtDNA does not have termination codons.

Mitochondrial Disease

• Mitochondrial dysfunction can lead to disorders, often linked to ageing or caused by mtDNA damage.
• Damage to mtDNA can lead to cellular dysfunction.
• Many cancers, or disorders, have been linked to mtDNA mutations.

Energy Failure through Damage

• Cells struggle to generate enough energy (ATP) if oxidative phosphorylation is compromised.
• They may shift to anaerobic respiration converting pyruvate to lactate.
• This results in cellular acidosis.

Test your knowledge on key concepts of cell biology, focusing on active transport mechanisms, DNA replication phases, and the role of genes. This quiz will challenge you with questions about facilitated diffusion, ion channels, and transcription processes. Engage with these essential topics to deepen your understanding of cellular functions.