Gene Splicing 9 Flashcards
What is the link between organism size and genome complexity
What is c-value paradox and what explains it
Eukaryotes have more that DNA that does not code for protein or for any other functional product molecule than prokaryotes.
size and organism complexity. Specifically, it refers to cases where organisms with relatively simple morphological or developmental features have genomes that are much larger and more complex than expected.
Traditionally, it was assumed that more complex organisms would have larger genomes because they would require more genes to encode the additional functions necessary for their complexity. However, this assumption doesn’t always hold true.
For example, some single-celled organisms may have genomes that are much larger than those of more complex multicellular organisms. This observation poses a paradox because it suggests that genome size is not directly correlated with organismal complexity.
There are several proposed explanations for the c-value paradox, including:
Genome duplication: Some organisms undergo whole-genome duplication events, where their entire genome is duplicated. Over time, this can lead to an increase in genome size without necessarily adding complexity.
Non-coding DNA: Much of the genome in complex organisms consists of non-coding DNA, which does not directly encode proteins. This non-coding DNA may serve regulatory functions or be remnants of transposable elements. The presence of large amounts of non-coding DNA can contribute to larger genome sizes without directly affecting organismal complexity.
Genome size regulation: The size of a genome may be influenced by various factors such as population size, mutation rates, and the effectiveness of selection. These factors can lead to variation in genome size among different species, regardless of their complexity.
Structural complexity: Genome size may not be the sole determinant of organismal complexity. Other factors such as gene regulation, protein interactions, and cellular processes may also contribute to the complexity of an organism.
How does Non-Coding DNA’ explains C-Paradox ?
What is C0t analysis + steps
Explain C0t analysis
Explain this C0t analysis graph
What are the different categories of eukaryotic dna in the genome
What are the different parts of a simple eukaryotic transcription unit
CAP-binding site is located upstream of the RNA-polymerase-binding site in the promoter. This increases the binding ability of RNA polymerase to the promoter region and the transcription of the genes
what is the role of polyA site in eukaryotic transcription unit
protects mRNA from degradation and enhances mRNA translation
Stops translation
Where does mRNA come from
What is gene topography *
it refers to the spatial organization and arrangement of genes within a genome or a particular chromosome
What are Functional” Repetitive Sequences
What are multigene families (an e.g of a family of coding gene)
What is a Dispersed Multigene Family (an e.g of a family of coding gene)
what are Non-functional” Repetitive Sequences
what are transposon*
A transposon is a DNA sequence that has the ability to move or transpose within a genome.
Transposons typically consist of two main components: the transposase gene and the terminal inverted repeats (TIRs). The transposase gene encodes the enzyme responsible for catalyzing the movement of the transposon. The TIRs are short DNA sequences found at both ends of the transposon, which serve as recognition sites for the transposase enzyme.
They can disrupt genes or regulatory sequences when they insert into coding or regulatory regions, leading to mutations or changes in gene expression. Transposons can also contribute to genome evolution by generating genetic diversity and promoting genomic rearrangements.
That are SINES and LINES
What is spacer DNA *
Spacer DNA refers to the non-coding DNA sequences found between genes or other functional elements within a genome. These regions typically do not contain coding sequences for proteins or functional RNA molecules. Spacer DNA can vary significantly in length and composition among different organisms.
Spacer DNA does not encode proteins or functional RNAs. Instead, it often consists of repetitive sequences, transposable elements, or sequences with regulatory functions, such as enhancers or insulators. (Role in regulation )
Regions of spacer DNA may loop out or interact with other regions, influencing the accessibility of genes to the transcriptional machinery.
What is the contour length of DNA*
The contour length of DNA refers to the total length of the DNA molecule if it were stretched out in a linear fashion, without considering its helical structure. It represents the distance between the two ends of the DNA molecule when it is fully extended.
The contour length of DNA can be calculated based on the number of base pairs (bp) it contains and the known length of one DNA base pair. In double-stranded DNA, one full turn of the helix corresponds to approximately 10 base pairs and spans about 3.4 nanometers in length. Therefore, the contour length (L) of double-stranded DNA can be calculated using the following formula:
L=Number of base pairs × Length of one base pair
Why do chromosomes need to be condensed
What experiment was carried out to test DNA packaging*
Isolation of Chromatin: Chromatin, which consists of DNA wrapped around histone proteins, was isolated from cells. This chromatin preparation retains the structural organization of DNA in the nucleus.
Treatment with Micrococcal Nuclease: The isolated chromatin was treated with micrococcal nuclease. This enzyme digests the DNA that is not tightly bound to histone proteins, leaving behind the DNA that is organized into nucleosomes.
Analysis of DNA Fragments: After digestion, the DNA fragments were analyzed using gel electrophoresis or other biochemical techniques. Gel electrophoresis separates DNA fragments based on their size, allowing researchers to visualize and analyze the distribution of DNA fragments resulting from nuclease digestion.
Interpretation of Results: The experiment revealed a pattern of DNA fragments corresponding to multiples of the DNA length protected by nucleosomes. This pattern of protected DNA fragments indicated that DNA was organized into repeating units, known as nucleosomes, which are composed of DNA wrapped around histone proteins.
What is a nucleosome* ( first stage of compaction of DNA)
A nucleosome is the basic structural unit of DNA packaging in eukaryotic cells. It consists of a segment of DNA wrapped around a core of histone proteins. Nucleosomes play a fundamental role in organizing and compacting the long strands of DNA, allowing it to fit within the nucleus of the cell.
What is a chromatosome*
A chromatosome is a structural unit of chromatin, the complex of DNA and proteins found in the nucleus of eukaryotic cells. It consists of a nucleosome, which is the fundamental repeating unit of chromatin, along with an additional histone protein known as histone H1.
What is a solenoid * (second stage of compaction)
Nucleosome Stacking: According to the solenoid model, nucleosomes are stacked upon each other in a helical or coiled arrangement, similar to the coils of a solenoid. This stacking allows for further compaction of chromatin
Which is a linker *
Linker DNA: The DNA segments that link adjacent nucleosomes, known as linker DNA, are thought to be relatively straight and flexible. They extend between adjacent nucleosomes within the solenoid structure
What is a scaffold association (3rd stage of compaction) *
Protein Scaffold or Matrix: The nucleus contains a network of proteins, often referred to as the nuclear matrix or nuclear scaffold, which provides structural support and organization to the chromatin. This protein scaffold is thought to play a role in anchoring specific DNA regions and organizing the spatial arrangement of chromosomes within the nucleus.
Scaffold Association: Scaffold association refers to the attachment or association of specific DNA segments or chromosomal regions with the protein scaffold or matrix. This association may be stable or dynamic, depending on the cellular context and the functional requirements of the genome.
What is a giant supercoil of DNA ( fourth stage of compaction)
Supercoiling is an essential mechanism for compacting the long DNA molecules into the small confines of the cell and for regulating DNA accessibility for processes such as transcription, replication, and repair.
What is the final stage of compaction of DNA
What is the structure of a chromosome
What is the role of a telomere of a chromosome *
Chromosome Protection: Telomeres protect the ends of chromosomes from degradation, fusion, and recombination. Without telomeres, the ends of chromosomes would resemble broken DNA strands, leading to activation of DNA damage response mechanisms and potential loss of genetic material.
Prevention of Chromosomal Fusion: Telomeres prevent the ends of different chromosomes from sticking together and forming aberrant structures called end-to-end fusions. This helps maintain the distinct identity of individual chromosomes and prevents genomic instability.
Facilitation of Chromosome Replication: During DNA replication, the enzyme complex responsible for replicating DNA, known as the DNA polymerase, cannot fully replicate the ends of linear chromosomes. This is because of the inability of DNA polymerase to synthesize DNA in a 5’ to 3’ direction at the very end of a linear template. Telomeres provide a solution to this problem by serving as a template for the synthesis of short repetitive sequences by the enzyme telomerase. This process, called telomere elongation, ensures that the ends of chromosomes are fully replicated and maintained during cell division.
Cellular Senescence and Aging: Telomeres undergo gradual shortening with each round of cell division due to incomplete replication and other factors. Eventually, when telomeres become critically short, cells enter a state of replicative senescence, where they stop dividing and may undergo programmed cell death (apoptosis). This process is thought to contribute to aging and age-related diseases.
Regulation of Gene Expression: Telomeres can influence gene expression and chromatin structure in the vicinity of chromosome ends through mechanisms such as telomere position effect (TPE). TPE involves the silencing of genes located near telomeres, which can impact cellular function and differentiation.
How is mRNA processed *
Capping
Splicing
Polyadenylation
What is RNA capping + why is it needed *
After transcription initiation, the pre-mRNA molecule undergoes capping, where a 7-methylguanosine cap is added to the 5’ end of the mRNA. This cap helps protect the mRNA from degradation and facilitates mRNA export from the nucleus to the cytoplasm. Additionally, the cap is essential for the initiation of translation.
What is polyadenylation and how does it occur
Polyadenylation is a post-transcriptional modification process in which a stretch of adenine nucleotides, known as a poly(A) tail, is added to the 3’ end of a pre-mRNA molecule. This modification plays essential roles in mRNA stability, export from the nucleus to the cytoplasm, and translation efficiency. Polyadenylation is a conserved process found in eukaryotic cells.
Here’s an overview of how polyadenylation occurs:
Recognition of the Polyadenylation Signal: The polyadenylation process is initiated by the recognition of specific sequence elements within the pre-mRNA molecule. One of the key elements is the polyadenylation signal sequence, which typically consists of the hexamer sequence AAUAAA (or a similar variant) located upstream of the site where polyadenylation will occur.
Cleavage and Polyadenylation Complex Formation: After transcription of the pre-mRNA, the polyadenylation signal sequence is recognized by a complex of proteins, known as the polyadenylation complex or polyadenylation machinery. This complex includes several protein factors, including cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), and poly(A) polymerase (PAP).
Cleavage of the Pre-mRNA: The polyadenylation complex cleaves the pre-mRNA molecule downstream of the polyadenylation signal sequence, typically between the signal sequence and a downstream GU-rich element. This cleavage reaction generates a free 3’ end on the pre-mRNA.
Polyadenylation: Following cleavage, the poly(A) polymerase (PAP) enzyme adds a stretch of adenine nucleotides (A’s) to the free 3’ end of the pre-mRNA. This process is template-independent, meaning that the poly(A) tail is synthesized without the need for a DNA template.
Poly(A) Tail Length Regulation: The length of the poly(A) tail can vary and is dynamically regulated in response to cellular signals and environmental cues. In general, longer poly(A) tails are associated with increased mRNA stability and translation efficiency.
What is the first step of slicing (of pre-mRNA )
What i s the second step of splicing
What is the spliceosome and what does it do *
The spliceosome is a complex of RNA and protein molecules found in the nucleus of eukaryotic cells. It plays a crucial role in the process of pre-mRNA splicing, which is the removal of introns and joining of exons to produce a mature mRNA molecule.
Composition: The spliceosome is composed of small nuclear ribonucleoproteins (snRNPs), which are complexes of small nuclear RNA (snRNA) molecules and associated proteins. The major snRNPs involved in splicing are U1, U2, U4, U5, and U6 snRNPs. Additionally, there are numerous non-snRNP proteins that associate with the spliceosome to facilitate its function.
Splicing Process: Pre-mRNA splicing involves two sequential transesterification reactions: splice site recognition and catalysis of phosphodiester bond formation. The spliceosome recognizes specific sequences at the exon-intron boundaries, including the 5’ splice site, the branch point sequence (located near the 3’ end of the intron), and the 3’ splice site. These sequences are recognized by base pairing interactions between the snRNAs and the pre-mRNA.
The spliceosome assembles onto the pre-mRNA through a series of dynamic and stepwise interactions. Initially, the U1 snRNP binds to the 5’ splice site, and the U2 snRNP binds to the branch point sequence. This assembly is followed by recruitment of the U4/U6.U5 tri-snRNP complex, forming the mature spliceosome.
Splicing Catalysis: Once assembled, the spliceosome undergoes conformational changes and rearrangements to bring the splice sites into close proximity and catalyze the splicing reactions. During the first step of splicing, the 5’ splice site is cleaved, and the 5’ end of the intron is covalently linked to the branch point sequence, forming a lariat intermediate. In the second step, the 3’ splice site is cleaved, and the exons are ligated together, releasing the intron in the form of a lariat structure.
What are snRNAs
What is alternative splicing
What are group I introns
What are group II introns *
Self-Splicing Activity: Group II introns are capable of catalyzing their own splicing reactions, leading to the removal of the intron sequence from a precursor RNA molecule and the joining of the flanking exon sequences. This self-splicing activity occurs through a series of coordinated RNA folding and conformational changes that bring together the splice sites and catalytic residues within the intron.
Secondary Structure: Group II introns have a complex secondary structure consisting of several conserved structural domains, including catalytic core domains and binding sites for cofactors such as magnesium ions. The secondary structure of group II introns is highly conserved across diverse organisms, suggesting a common evolutionary origin.
Mechanism of Splicing: The splicing mechanism of group II introns involves two transesterification reactions that occur in two steps, similar to the spliceosome-mediated splicing of nuclear pre-mRNA. In the first step, the 2’ hydroxyl group of an internal adenosine nucleotide within the intron attacks the 5’ splice site, resulting in cleavage of the phosphodiester bond and formation of a lariat intermediate. In the second step, the 3’ hydroxyl group of the 5’ exon attacks the 3’ splice site, leading to exon ligation and release of the intron lariat.
What is the purpose of introns
