lecture 1 Flashcards
explain and detail the learning outcomes
- Explain why there is a need to maintain high sequence similarity during replication:
Maintaining high sequence similarity during DNA replication is essential because:
Genetic Fidelity: Accurate replication ensures that the genetic information passed from one cell generation to the next remains consistent. This is vital for preserving the structure and function of proteins encoded by the DNA.
Prevention of Mutations: Errors in replication can introduce mutations, which may lead to nonfunctional or harmful proteins, potentially causing diseases such as cancer.
Evolutionary Conservation: Many DNA sequences are conserved due to their critical biological functions. High-fidelity replication ensures these important sequences are preserved across generations and species.
- Identify key enzymes needed for replication that are common to both eukaryotes and prokaryotes and explain their function:
DNA Helicase: Unwinds the DNA double helix by breaking the hydrogen bonds between the complementary strands, creating the replication fork.
DNA Polymerase: Synthesizes the new DNA strand by adding nucleotides to the 3’ end of the growing strand, using the original strand as a template. It also proofreads to ensure accuracy.
Primase: Synthesizes short RNA primers to provide a starting point for DNA polymerase to begin replication.
Ligase: Joins Okazaki fragments on the lagging strand by sealing nicks in the sugar-phosphate backbone.
Single-Strand Binding Proteins (SSBs): Bind to single-stranded DNA to prevent reannealing and protect the strands during replication.
Topoisomerase: Relieves supercoiling and tension ahead of the replication fork by cutting and rejoining DNA strands.
These enzymes ensure the process of replication is fast, efficient, and accurate across both prokaryotic and eukaryotic organisms. - Describe the steps of replication and explain where in this process there is an opportunity for a loss of sequence conservation:
Steps of Replication:
Initiation:
Helicase unwinds the DNA, and primase lays down RNA primers.
Elongation:
DNA polymerase extends the new strand by adding complementary nucleotides to the template strand.
Leading strand is synthesized continuously, while the lagging strand is synthesized in Okazaki fragments.
Termination:
Replication is completed when DNA polymerase reaches the end of the template or when two replication forks meet.
Opportunities for Loss of Sequence Conservation:
Errors in DNA Polymerase Activity: Despite proofreading, DNA polymerase can make errors, leading to mismatches that can introduce mutations if not corrected.
Replication of Repetitive Sequences: Repetitive sequences (e.g., microsatellites) are prone to slippage, leading to insertions or deletions.
Mutagenic Factors: Environmental mutagens or internal factors like reactive oxygen species (ROS) can damage DNA during replication, leading to incorrect base pairing. - Apply principles of biochemistry to explain under what circumstances DNA sequences can be good and when it can be bad (using examples might help you):
Beneficial DNA Sequences:
Functional Genes: DNA sequences that encode functional proteins, such as the insulin gene, are crucial for health and proper cellular function.
Evolutionary Adaptations: Mutations that provide a survival advantage can be beneficial. For example, a mutation that leads to antibiotic resistance in bacteria may be advantageous in the presence of antibiotics.
Harmful DNA Sequences:
Harmful Mutations: DNA mutations that alter the function of important proteins can lead to diseases. For instance, a single nucleotide mutation in the beta-globin gene can cause sickle cell anemia.
Oncogenes: Mutations in certain genes (e.g., RAS or TP53) can lead to uncontrolled cell growth, resulting in cancer.
Summary:
High Sequence Similarity: Necessary for the accurate transfer of genetic information.
Common Enzymes: DNA polymerase, helicase, and primase are key in both prokaryotic and eukaryotic replication.
Replication Process: Initiation, elongation, and termination stages provide opportunities for errors.
Biochemistry and DNA: DNA sequences are “good” when they contribute to normal function and “bad” when they lead to detrimental mutations or diseases
What do you remember about DNA replication?, detail the fundamentals
- during semi conservative replication the 2 new DNA strands both contain half of the original DNA (in the form of half the original strand)
- DNA replication occurs during the S phase of the cell cycle
- DNA replication is carried out by sigma factors in prokaryotes- Sigma factors recognize specific
DNA sequences called promoters, typically located just upstream of the genes they control. Different sigma factors recognize different promoter sequences, ensuring that the right set of genes is expressed under specific conditions.
Sigma factors are proteins that are part of bacterial RNA polymerase (RNAP) and are responsible for the initiation of transcription:
Function
Sigma factors help RNAP recognize promoters, open them, and separate DNA strands. They also help with the first steps of RNA synthesis.
Specificity
Sigma factors determine which promoter DNA binds to, which allows for the efficient initiation of transcription
-there are similar enzymes required to carry out DNA replication in prokaryotes and eukaryotes
Why do eukaryotes use semi-conservative replication?
in order to maintain integrity, and high fidelity of the DNA sequence- eukaryotes have very large genomes with lots of regions of non-coding DNA which is important for the regulation of transcription- low fidelity can be fatal
Why Integrity of the DNA is essential
Inheritance of germline sequences helps to introduce genetic variation among a population while there are also mechanisms in place to limit errors in replication that lead to a change in the germline DNA
-changes in nuclei acid sequence can result in changes of the amino acid sequence- this can affect the formation of proteins and their shapes
–changes in amino acid sequence may not affect the type of protein formed
–however changes in amino acid sequence may also result in a change to the shape of the protein, change in the protein function or change in the expression of the protein
- a possible change that can occur to change the shape of a protein is a change from a specific amino acid to one with a different charge within the amino acid sequence- this can for example result in amino acids repelling or attracting one another in the sequence which can ultimately affect the proteins shape as a result the formed protein may no longer be able to bind to a substrate that it was required to bind to
- ergo the change in amino acid sequence can mean the function of the protein is to be affected or the level of expression of the protein is affected because if it cannot fold properly the protein will be exported from the ER and degraded
- these potential changes can be passed down through generations if not fixed and result in diseases when expressed as a potential phenotype
potential changes in the DNA sequences at the gamete and zygote level can create detrimental effects as potential diseases may be passed on to the next generation
Mechanisms for the preservation of DNA integrity
*Presence of a nuclear envelope
*Condensation of chromatin when genes are not active- prevents unwanted things from accessing DNA due to high level of compacting around histones
*Semi-conservative replication mechanism- reduces the likelihood of changes to base pairing of DNA
*-Presence of chromosomal structures such as:
*Telomeres –repeating sequences of DNA that are shortened with age, they are replicated by a different mechanism to other parts of the chromosome
*Centromere –repetitive sequences of DNA that are highly packaged to provide a robust attachment site to the mitotic spindleAlberts
why may changes to the amino acid sequence have no affect on the protein formation?
Changes to the amino acid sequence of a protein, known as mutations, do not always affect protein formation or function due to several factors:
- Genetic Code Degeneracy (Redundancy)
The genetic code is degenerate, meaning that multiple codons (three-nucleotide sequences in DNA or RNA) can code for the same amino acid.
For example, the amino acids leucine, serine, and arginine each have six different codons that encode them.
If a mutation occurs in the DNA sequence that changes a codon to another codon for the same amino acid (a synonymous mutation or silent mutation), the amino acid sequence of the protein remains unchanged, and the protein’s structure and function are not affected. - Similar Amino Acid Properties (Conservative Substitution)
Even when a mutation changes an amino acid, the substituted amino acid may have similar properties to the original one.
For example, if a hydrophobic amino acid like valine is replaced by another hydrophobic amino acid like leucine, the overall structure and function of the protein may remain largely unaffected because both amino acids have similar chemical properties.
These types of substitutions are called conservative substitutions, as they do not significantly disrupt the protein’s folding or activity. - Location of the Mutation
The effect of a mutation depends on where in the protein the change occurs:
Non-critical regions: If the mutation occurs in a non-essential part of the protein, such as a loop or an area that is not directly involved in the protein’s function, the protein might still fold and function normally.
Active or binding sites: Mutations in the active site of an enzyme or a binding site of a receptor are more likely to have a significant impact on protein function. - Protein Folding and Structure
Proteins have a robust ability to fold into their functional structures due to the thermodynamic stability of their native state.
Small changes in the amino acid sequence might not be enough to destabilize the protein’s 3D structure, allowing it to fold properly and maintain its function.
Chaperone proteins in cells also assist in the correct folding of proteins, helping to prevent potential issues caused by minor mutations. - Redundant and Compensatory Pathways
Some proteins are part of larger protein families or biochemical pathways with redundant functions.
If a mutation affects one protein’s function, other proteins or pathways might compensate for its loss or reduced activity, minimizing the impact of the mutation on cellular functions.
Summary
Changes to the amino acid sequence may have no effect on protein formation or function due to the degeneracy of the genetic code, conservative amino acid substitutions, the mutation’s location within the protein, the protein’s inherent stability, and compensatory cellular mechanisms. These factors allow proteins to tolerate some level of variation without compromising their structure or activity.
what is The Nuclear Envelope made up of?
➢Consists of two concentric membranes
➢Contiguous with one another but are functionally distinct
➢To gain entry into the nucleus proteins and nucleic acids must be transported through the nuclear pore comple
how does the nuclear pore complex work?
how does the nuclear pore complex
The nuclear pore complex (NPC) is a large protein structure embedded in the nuclear envelope that regulates the transport of molecules between the nucleus and the cytoplasm in eukaryotic cells. The NPC acts as a gateway, controlling the exchange of materials, such as RNA, proteins, ions, and signaling molecules, to ensure that only the appropriate molecules move in and out of the nucleus.
Structure of the Nuclear Pore Complex
The NPC is a large protein assembly made up of multiple nucleoporins (proteins that make up the pore).
It has a symmetrical structure with eightfold radial symmetry and consists of multiple parts:
Cytoplasmic filaments: Extend into the cytoplasm and play a role in recognizing and interacting with transport molecules.
Central channel: The main pore through which molecules pass.
Nuclear basket: Extends into the nucleus and aids in the selective transport of cargo.
Ring structures: Found on both the nuclear and cytoplasmic sides, providing the framework for the NPC.
How the NPC Works: Transport Mechanisms
The transport through the NPC can be classified into two types: passive diffusion and active transport.
Passive Diffusion:
Small molecules and ions (less than 40 kDa in size) can passively diffuse through the NPC without the need for energy or specific transport signals.
This process is non-selective, allowing molecules to move through the central channel freely based on their size and concentration gradient.
Active Transport:
Large molecules like RNA, proteins, and ribonucleoprotein complexes (greater than 40 kDa) require active transport mechanisms to pass through the NPC.
Active transport requires:
Nuclear Localization Signals (NLS): For proteins that need to be imported into the nucleus. NLS is a specific sequence of amino acids on the cargo protein that signals it should be transported into the nucleus.
Nuclear Export Signals (NES): For molecules that need to be exported out of the nucleus to the cytoplasm.
Transport receptors (importins and exportins) recognize these signals and bind to the cargo molecules to facilitate their movement through the NPC.
Steps of Active Transport through the NPC
- Cargo Recognition:
In the cytoplasm, transport receptors like importins bind to the cargo protein containing an NLS.
This binding forms a cargo-receptor complex.
- Translocation through the NPC:
The cargo-receptor complex moves through the NPC, interacting with the nucleoporins that line the channel.
These interactions are facilitated by FG repeats (phenylalanine-glycine-rich regions) present in the nucleoporins, which act as binding sites for transport receptors.
- Release and Cargo Delivery:
Once inside the nucleus, the complex encounters Ran-GTP, a small GTPase that triggers the release of the cargo from the receptor.
The receptor, now bound to Ran-GTP, is transported back to the cytoplasm.
In the cytoplasm, Ran-GTP is converted to Ran-GDP, causing the receptor to release Ran and become ready to bind to new cargo.
- Recycling:
The transport receptors are continuously recycled between the nucleus and the cytoplasm, making the process efficient and energy-effective.
Regulation of Transport through the NPC
Ran-GTP gradient: The asymmetric distribution of Ran-GTP in the nucleus and Ran-GDP in the cytoplasm is critical for the directionality of transport.
Post-translational modifications: Changes in the state of nucleoporins or cargo proteins can regulate transport rates.
Cell cycle control: During different phases of the cell cycle, the activity and number of NPCs can change, affecting nuclear transport.
Importance of the Nuclear Pore Complex
Gene expression regulation: NPCs control the export of mRNA from the nucleus to the cytoplasm, which is essential for protein synthesis.
Signal transduction: They allow signaling molecules to enter the nucleus and affect gene regulation.
Protection of genetic material: By controlling what enters and leaves the nucleus, the NPC ensures the integrity and proper functioning of the cell’s genetic material.
Summary
The nuclear pore complex acts as a selective gatekeeper for the movement of molecules between the nucleus and cytoplasm. It uses passive diffusion for small molecules and active transport for larger molecules through the interaction of transport receptors with specific nuclear localization and export signals. This mechanism is crucial for maintaining cellular functions such as gene expression, signal transduction, and the overall integrity of the cell’s genome.
what are the components of chromatin?
Components of chromatin
*Two molecules of each of four histone (H2A, H2B, H3and H4) make an octamer.
*Histones are very well conserved between species
*146 ntof DNA winds 1.65 times around histone core to form the nucleosome.
*Hydrogen bonds form between the DNA and the histone octamer
*Each nucleosome is separated from the next by up to 80 ntof linker DNA
*Histone H1 works as a clamp
linker DNA is The double-stranded DNA that joins the DNA of one nucleosome to the next. Linker DNA is usually about 20 base pairs in length, but can vary between organisms and cell types. It helps to stabilize the structure of chromatin by associating with a linker histone H1.
why can DNA packaging prevent enzymes from gaining access to it?
As packaging gets more complex the “room” taken up by DNA gets smaller and it is harder for proteins to gain access to the DNA to “read” it (transcription) or replicate it (replication)
how is Replication is based upon the formation of base-pairs
Replication is based upon the formation of base-pairs
*Replication must occur before cell division starts.
*The original copy is used as a template to complimentary copy of genomic DNA.
*This occurs in a stepwise manner:
1.The double helix must be separated into 2 strands, generating the replication fork (separation).
2.DNA Primase makes short RNAs (primers) acting as a platform for DNA polymerase (Initiation –leading strand only).
3.The template strand bases are recognised by free bases and then joined together by DNA polymerase (polymerization)
4.On the lagging strand DNA primase creates primers every 100-200 bp, which are extended by DNA polymerase and joined together by DNA ligase
what enzymes are involed in DNA replication?
DNA helicase
DNA primase
DNA ligase
DNA polymerase
Topoisomerase
Step 1a: Separation
Before replication can begin, the helix needs to be separated into 2 template strands by DNA helicase.
DNA helicase binds to a single strand of DNA and spins around it, propelled by the hydrolysis of ATP, physically breaking the hydrogen bonds between bases.
Step 1b: Separation
Once separated the single stranded DNA is bound by single stranded binding (SSB) proteins to prevent secondary structures forming
Step 2: Initiation
Once the replication fork is formed DNA Primasemakes short RNA primers of around 10nt in length which recognise bases at the replication fork