Module 3 Flashcards
Describe the key features of group 1 intron splicing
Self-splicing (in vitro, need proteins to fold the RNA into the catalytically active structure in vivo)
Requires an external guanosine cofactor to splice
Two transesterification reactions
Proteins required for splicing are encoded by the introns or other genes
RNA tertiary structure forms a molecular cradle that keeps everything together while this occurs
Describe the key features of group 2 intron splicing
Also self-splicing (in vitro, need proteins to fold the RNA into the catalytically active structure in vivo),
Requires a bulged A
Creates an intron lariat with a 2’-5’ phosphodiester bond between A and G
Describe the key features of archaeal intron splicing
Spliced by an endonuclease protein, removed by a cut-and-rejoin mechanism.
Requires ATP, an endoribonuclease, and a ligase.
Intron transcripts generate a bulge-helix-bulge motif at the exon-intron junction, which is recognized by the splicing endoribonuclease, which cuts within the three-nucleotide bulges at symmetric positions.
Exons are ligated by a specific ligase. Sometimes structural rearrangement occurs post-ligation
Describe the key features of nuclear tRNA intron splicing
Cleaved by endonuclease and then sealed by ligase.
There are 2 classes of nuclear tRNA genes, distinguished by either the presence or absence of an intron (in humans, only tyrosine and leucine charged tRNA contain introns).
Two main reactions in the splicing mechanism. 1) the intron-containing pre-tRNAs are cleaved by a tRNA splicing endoribonuclease at the 5’ and 3’ boundaries, freeing the paired tRNA halves. 2) the paired halves are joined by tRNA ligase in an ATP and GTP dependent reaction, which leaves a 2’-phosphate at the 3’-OH splice junction
Describe the key features of nuclear pre-mRNA intron splicing
Removed by the spliceosome during RNA processing, also has 2 transesterification reactions and an intron lariat.
Spliceosome has 5 uracil-rich snRNPs (U1, U2, U4, U5, U6).
Processing is coupled to transcription.
Splicing machinery is recruited cotranscriptionally by directly interacting with the RNA polymerase II CTD.
Basic two-step transesterification reactions are similar to group 2 introns, but pre-mRNA introns are not self-splicing and can’t fold themselves into the proper secondary and tertiary structures. They need the spliceosome, a protein-directed ribozyme, to generate the 3D structure necessary for splicing.
Describe the key features of the composition and function of the spliceosome.
The spliceosome is a large complex that removes introns during RNA processing. It consists of 5 uracil-rich snRNPs (U1, U2, U4, U5, U6) and about 150 proteins, that include those that make up the protein ring in the center and those that surround and support the catalytic active site. It is a protein directed ribozyme.
The spliceosome generates the 3D structure required for nuclear pre-mRNA splicing.
Aside from the spliceosome, the nuclear pre-mRNA splicing pathway looks identical to the group 2 intron splicing pathway (bulged adenosine, two transesterification reactions, 2’-5’ phosphodiester bond, lariat intron)
The spliceosome is a protein-directed ribozyme. Its protein components surround and support its catalytic RNA active site (active sites are the U2, U6, and U5 snRNA components).
Does mRNA polyadenylation occur before or after termination of transcription by RNA polymerase II?
After, when mRNA is released and cleaved
Why is it said that alternative splicing is a mechanism for generating protein diversity from a small set of genes?
It’s a way to make many different proteins from monocistronic genes. Prokaryotes + shine-dalgarno sequences + internally binding ribosomes versus eukaryotes + 5’-methylguanosine cap + non-internally binding ribosomes.
Alternative splicing provides a versatile means of regulating gene expression while contributing to protein diversity by expanding the proteome. Alternative splicing in the coding regions of DNA creates novel proteins, and alternative splicing in the untranslated regions allows for novel translational regulation, stability, and mRNA localization expressions (remember that mRNA from the UTRs is often involved in regulating transcription, providing stability, and mRNA localization, even though it’s not coded into a protein)
Describe the key features of RNA editing in trypanosomes
RNA editing discovered in mitochondrial RNA.
Adding Us can correct internal frameshifts, create start or stop codons, or create orfs.
Editing is controlled by gRNAs transcribed for by DNA.
The gRNAs are complementary to the mature mRNAs in the edited region. gRNAs provide specificity by complementary base pairing to the mature mRNA. E
diting occurs 3’ → 5’ and requires multiple gRNAs. Editosome catalyzes editing.
Remember that G can pair with U in RNA!
Describe the key features of RNA editing in longfin squid
ADAR2, A →I editing in which Inosine is read as Guanosine by the translation machinery so some codons are re-coded and some protein functions may change because of it.
Result is proteome diversification that allow squids to rapidly adapt to changes in their environment.
Describe the key features of RNA editing in ApoB RNA editing in humans
C → U editing. ApoB has a role in the assembly, transport, and metabolism of plasma lipoproteins.
Spliceosome removes the introns.
The editing complex confers specificity using the APOBEC1 complementation factor (ACF) subunit of the RNA-specific cytidine deaminase that carries out the recognition and catalysis of ApoB RNA editing. The ACF subunit is an RNA-binding protein and recognizes an 11-nucleotide “mooring sequence” just downstream of the edited C in the ApoB mRNA.
siRNA
One of the two major classes of RNAi. siRNAs trigger the formation of RISC.
These guys are auto-silencing, meaning that they silence the same or similar locus to that which they originated from.
They are found in viruses, TEs, heterochromatin, exogenous genes or dsDNA inserted into the gene by a scientist.
Defence of the genome.
miRNA
One of the two major classes of RNAi.
miRNAs are short RNA molecules that fold into a hairpin to create a dsRNA that then triggers the RNAi machinery. They regulate the expression of most protein-coding genes.
These guys are hetero-silencing, meaning that they are derived from unique genes and specify the silencing of very different ones.
They are processed from gene transcripts that form hairpin structures, and are often found located within the introns of protein-coding mRNA.
They are part of post transcriptional gene regulation as they target mRNAs for degradation.
Describe the base-pairing interactions that occur in translation
tRNAs add different bases to the mRNA strand in the ribosome.
The tRNAs carry the amino acid building blocks and assemble the protein by base pairing with the appropriate mRNA codon in the ribosome. The mRNA codes for the protein produced.
Describe the base-pairing interactions that occur in RNAi
dsRNAs, either siRNA or miRNA, interacts with the mRNA.
Sequence-specific gene silencing. Argonaute binds to one strand of the dsDNA and then uses it to find the complementary sequence on the mRNA, which is degraded.
Describe the base-pairing interactions that occur in RNA editing in trypanosomes
gRNA confers binding specificity by complementary base pairing with the mature mRNA in the editing region, this allows the RNA editing (insertion or deletion of Us) to occur
Describe the role of rRNA in protein synthesis
Forms a catalytic center, decoding site, A, P, and E sites, and the intersubunit interface. Put simply, it is the ribosome, and it holds everything together.
Describe the role of tRNA in protein synthesis
Works with the ribosome; pairs with complementary codons and delivers the next amino acid to the growing polypeptide chain.
Describe the role of mRNA in protein synthesis
Carries the genetic information required to create the proteins from the DNA in the nucleus to the ribosome. Has codons that pair with tRNA to translate code into amino acids.
What is an example of an RNA-protein interaction that influences the catalytic activity of a ribozyme?
Remember that ribozymes are RNA enzymes that self cleave, meaning they can act only once, unlike regular enzymes which can be recycled ad nauseu.
Spliceosomes are protein-directed ribozymes. Its protein components surround and support its catalytic RNA active sites (active sites are the U2, U6, and U5 snRNA components)
What effect does phosphorylation have on the function of eukaryotic initiation factor 2 (eIF2)?
Phosphorylation (remember that kinases phosphorylate) of elF2 blocks ternary complex formation, preventing translation.
What is “knockdown”?
Knockdown experimentation is used to study the effects of cis-regulatory elements, such as the promoter region or other upstream regulatory sequences, to see if their modification changes expression levels or inactivates the real target gene.nThe knockdown targeting sequence disrupts endogenous upstream regulatory elements while keeping the endogenous coding region intact. The target gene is never touched in knockdown experimentation, only the regulatory elements around it.
What are “knockouts”?
Mrs. Ritchie
Fr tho, they’re when a protein-coding gene (not a promoter or regulatory sequence) is removed or inactivated, halting all protein expression.
Distinguish between the terms “knockdown” and “knockout” with respect to analyzing gene function.
Knockdown reduces gene expression, while knockout turns the gene off completely.
Describe the key steps in transgenic technology
Pronuclear injection to insert a transgene into a fertilized egg. Integration into the fertilized egg’s genome is random.
Describe the key steps in gene targeting
Homologous recombination of targeting vector in embryo-derived stem cells is selected for, and then injected into a blastocyst-stage embryo. Disrupts or mutates the targeted gene, which can be used to knockin or knockout a gene.
Describe the key steps in gene editing
Precise editing of the targeted genome using CRISPR-Cas9
CRISPR-Cas system is used to precisely edit any cell type.
Describe the key steps in cloning by nuclear transfer
A genetically-identical organism created by nuclear transfer from the adult somatic cells to an egg
Method is to transfer the entire nucleus from the donor into an eunucleated, unfertilized egg. This creates (mostly) genetically identical individuals.
Does a cloned organism and its clone have identical nuclear DNA profiles?
Yes. All nuclear DNA in the cell comes from the donor, and all the egg’s chromosomes are removed from the egg during metaphase.
Does a cloned organism and its clone have identical mitochondrial DNA profiles?
No. Half of the mitochondrial DNA would come from the original Smarty, and the other half from the egg. This is because we leave the mitochondrial DNA in the egg.
Describe the underlying principles for DNA typing (DNA profiling/DNA fingerprinting) using short tandem repeat (STR) analysis and multiplex PCR.
Analyze polymorphic markers, which are unique biological identifiers that follow simple Mendelian patterns of inheritance, to match an individual with their DNA. Everyone inherits two of each STR one from the egg and the other from the sperm.
The basis of DNA typing is analysis of polymorphisms. The goal is to calculate a probability that only one person could have the same profile of markers. A variety of analytic techniques are used for this, including minisatellite analysis, PCR-based analysis, STR analysis, mtDNA analysis, Y chromosome analysis, and random amplification of polymorphic DNA (RAPD) analysis.
Only about 0.1% of the human genome differs from one person to another. The majority of the human genome represents intergenic DNA, regions that aren’t controlled by selection pressures, where mutations are usually maintained and transmitted from generation to generation.
Short tandem repeats (STRs) are variable regions that forensic geneticists like to use because they can generate the profile of the individual without giving information about the individual’s phenotypic traits.
STR analysis provides higher discriminating power than minisatellite analysis while requiring a smaller DNA sample size. STRs are widely distributed throughout the human genome. STR analysis is usually combined with a multiplex PCR reaction (multiplex PCR enables simultaneous amplification of many targets of interest in one reaction by using more than one pair of primers). The preferred technique for separating the STR loci is multichannel capillary electrophoresis, however regular gel electrophoresis can be used as well.
For multiplex PCR analysis of STRs, one primer in each pair is labeled with a different-colored fluorescent tag. The PCR amplification products are then mixed with labeled internal lane standards and detected using an automated sequencer. Different STR loci and alleles are separate by size based on their migration rate in the sequencer gel matrix and detected by color after laser-induced excitation of the fluorescent dyes
