Genetic Test #2
What are the steps that comprise eukaryotic translation initiation?
During initiation, the small ribosomal subunit binds to the start of the mRNA sequence. Then a transfer RNA (tRNA) molecule carrying the amino acid methionine binds to what is called the start codon of the mRNA sequence. The start codon in all mRNA molecules has the sequence AUG and codes for methionine. Next, the large ribosomal subunit binds to form the complete initiation complex.
What are the steps that comprise eukaryotic translation termination?
During the elongation stage, the ribosome continues to translate each codon in turn. Each corresponding amino acid is added to the growing chain and linked via a bond called a peptide bond. Elongation continues until all of the codons are read
What are the steps that comprise eukaryotic translation elongation?
During the elongation stage, the ribosome continues to translate each codon in turn. Each corresponding amino acid is added to the growing chain and linked via a bond called a peptide bond. Elongation continues until all of the codons are read.
What is the overall structure of a eukaryotic ribosome?
Eukaryotic ribosomes have two unequal subunits, designated small subunit (40S) and large subunit (60S) according to their sedimentation coefficients. Both subunits contain dozens of ribosomal proteins arranged on a scaffold composed of ribosomal RNA (rRNA). A P site and E(exit) site
How do base excision and nucleotide excision repair mechanisms differ?
Excision repair: Damage to one or a few bases of DNA is often fixed by removal (excision) and replacement of the damaged region. In base excision repair, just the damaged base is removed. In nucleotide excision repair, as in the mismatch repair we saw above, a patch of nucleotides is removed.
How are mRNAs degraded?
Fragments of mRNA generated by RISC (Fig. 10.45) cleavage become degraded by the general mRNA degradation machinery. Most mRNA degradation occurs using the CCR4/Not complex, targeted to the 3′UTR of the mRNA and directed by elements and proteins binding to this part of the mRNA. Increasing Stability: While cells degrade messenger RNA to regulate the amount of proteins that can be translated from each mRNA molecule, they also modify mRNA molecules in a way that increases the stability of the molecule and increases the protein output under specific conditions and at certain times.
What are the two transesterification reactions that underlie pre-mRNA splicing?
From a biochemical viewpoint, the RNA splicing reaction is a relatively simple process that consists of two transesterification reactions 1) 2' OH branch site (A) attacks 5' splice site (phosphoryl group of G) and forms 3 way junction 2) 3' OH of newly spliced 5' exon attacks the 3' splice site and releases the lariat!
Do excised introns have any functions?
Functions Associated with Excised Introns. Once an intron had been excised, it typically becomes part of post-splicing complexes that lead to efficient debranching and degradation But when an RNA gene is embedded within the intron, it is expressed upon intron removal, and outlives its intronic host.
. Why does a replication fork contain DNA helicase and single-stranded DNA binding proteins?
Helicaseunwinds the helix, and single-strand binding proteins prevent the helix from re-forming. Topoisomeraseprevents the DNA from getting too tightly coiled ahead of the replication fork. •DNA Helicase separates the two strands •Single-stranded DNA binding proteins keep strands separate until they are copied •RNA primers are ~20 ntslong & synthesized by primase (specialized RNA polymerase)
What are the cell cycle phases of a eukaryotic cell?
In eukaryotes, the cell cycle consists of four discrete phases: G1, S, G2, and M. The S or synthesis phase is when DNA replication occurs, and the M or mitosisphase is when the cell actually divides.
What are mutagens?
In genetics, a mutagen is a physical or chemical agent that changes the genetic material, usually DNA, of an organism and thus increases the frequency of mutations above the natural background level.
Why are RNA primers necessary for DNA replication?
In living organisms, primers are short strands of RNA. ... The synthesis of a primeris necessary because the enzymes that synthesize DNA, which are called DNApolymerases, can only attach new DNA nucleotides to an existing strand of nucleotides. The primer therefore serves to prime and lay a foundation for DNAsynthesis. DNA polymerase can only add dNTPs to a pre-existing 3' end called a primer. The primers are short RNAs that are subsequently removed & replaced with DNA
What specific steps are involved in capping & polyadenylation and when do they occur relative to transcription?
In nuclear polyadenylation, a poly(A) tail is added to an RNA at the end of transcription. On mRNAs, the poly(A) tail protects the mRNA molecule from enzymatic degradation in the cytoplasm and aids in transcription termination, export of the mRNA from the nucleus, and translation. Capping is a three-step process that utilizes the enzymes RNA triphosphatase, guanylyltransferase, and methyltransferase. Through a series of three steps, the cap is added to the first nucleotide's 5' hydroxyl group of the growing mRNA strand while transcription is still occurring. The answer is the last option : it occurs before pre management reaches a chain length of 30 nucleotides. The capping occurs to protect the mRNA from degrading during the transport out of nucleus and translation. This occurs only in the eukaryotic cells. The cap includes the 7-methylguanosine added to the first nucleotide of the mRNA with 5'-5' glycolic bond.
What are the functional regions of a typical eukaryotic mRNA and what post-transcriptional, translational, and even post-translational events, do they regulate?
In particular, mRNA untranslated regions are involved in many post-transcriptional regulatory pathways that control mRNA localization, stability and translation efficiency. The first one involves the control of transcription mediated by cis-acting DNA elements such as promoters, enhancers, locus control regions and silencers to produce a mature mRNA. mRNA UTRs known to mediate a similar post-transcriptional regulatory activity (e.g. same subcellular localization or stability of the mRNA) or belonging to genes sharing common features (e.g. genes involved in the cellular cycle, etc.). In this case, more sophisticated pattern discovery algorithms should be used, allowing a limited amount of pattern degeneracy (i.e. point mutations, insertions and deletions), which better reproduces structural features of known functional oligonucleotides. These methods, that can be roughly grouped into two categories, pattern-driven or sequence-driven
How can exons be "skipped" to generate alternatively spliced mRNAs?
Introns exist to mske it possible to assemble genes from various exon building blocks that encode modules of protein function, this type of assembly would allow the shufflig of exons to make new genes Alternative splicing of exons in a pre-mRNA transcript is an important mechanism which contributes to protein diversity in human. Splcing may occur b/n the splice donor site of one intron and the splice acceptor of a differnet intron downstream. Such alternative pslicing [roduces differnet mRNA molecules that may encode related proteins w/ differemt though partially overlapping AA sequences and functions, and also can cause the nucleotide seqeunce to roduce more than one type of polypeptide
How can a drug that causes the ribosome to ignore nonsense codons (premature termination codons) potentially be used to treat numerous, unrelated genetic diseases?
Premature termination codons (PTCs) in the coding regions of mRNA lead to the incorrect termination of translation and generation of non-functional, truncated proteins. Translational readthrough of PTCs induced by pharmaceutical compounds is a promising way of restoring functional protein expression and reducing disease symptoms, without affecting the genome or transcriptome of the patient. While in some cases proven effective, the clinical use of readthrough-inducing compounds is still associated with many risks and difficulties. Premature termination codons An extensive meta-analysis study, based on the Human Gene Mutation Database, has revealed that 12% of all described gene lesions causing human inherited diseases is caused by nonsense mutations (Mort et al., 2008). These mutations, by changing an amino acid coding triplet into a stop codon, introduce premature termination codons, PTCs, into the protein-coding gene sequence. PTCs may also be caused by other types of mutations, such as frameshifts (insertion or deletion other than multiple-of-three base pairs) or mutations in the conserved splice-site sequences (leading to a defective intron removal from the pre-mRNA)
What are the primary components involved in protein synthesis and their individual functions?
Protein synthesis is the process in which cells make proteins. It occurs in two stages: transcription and translation. Transcription is the transfer of genetic instructions in DNA to mRNA in the nucleus. It includes three steps: initiation, elongation, and termination. The three roles of RNA in protein synthesis. Messenger RNA (mRNA) is translated into protein by the joint action of transfer RNA (tRNA) and the ribosome, which is composed of numerous proteins and two major ribosomal RNA (rRNA) molecules.
How does Topoisomerase resolve the problem created when replication forks collide?
Relax DNA as opening up, making single stranded bits and pulling it apart, need to coil much tighter which will make DNA tangle over itself Need topimsimerase, break DNA apart amdallow other strand to go through it and puts it back together as the way it was before
What distinguishes "Leading" & "Lagging" strand synthesis?
Remember, since DNA replication proceeds bidirectionally from the origin: •The leading strand with left moving fork is the lagging strand with right moving fork •The lagging strand with left moving fork is the leading strand with right moving fork The leading strand is a single DNA strand that, during DNA replication, is replicated in the 3' - 5' direction (same direction as the replication fork).
How do we know that only the anticodon on a tRNA & not the amino acid it carries determines which amino acid will be incorporated during translation elongation?
Researchers can subject a charged tRNA to chemical treatments tht without altering the structure of tRNA, changes the AA it carriess One treatment replaces cytesine cattrird by tRNA with alanine, when this is added the system incorperates alenine in the polypeptide where mRNA contains a cyseteibe codon complementary to the anticodon of the tRNA cysetine
Why is semiconservative replication the most plausible model for DNA synthesis based on its structure?
Semiconservative replication describes the mechanism of DNA replication in all known cells. It derives its name from the production of two copies of the original DNA molecule, each of which contains one original strand, and one newly-synthesized strand. [1][2] The structure of DNA (as deciphered by James D. Watson and Francis Crick in 1953) suggested that each strand of the double helix would serve as a template for synthesis of a new strand. However, it was not known how newly synthesized strands combined with template strands to form two double helical DNA molecules. The semiconservative model of replication seemed most reasonable since it would allow each daughter strand to remain associated with its template strand.
What are the structural & functional features of tRNA?
Short, single stranded RNA molecules, 74-95 nucleotides long The tRNA molecule has a distinctive folded structure with three hairpin loops that form the shape of a three-leafed clover. One of these hairpin loops contains a sequence called the anticodon, which can recognize and decode an mRNA codon. Each tRNA has its corresponding amino acid attached to its end.
What distinguishes silent, nonsense, missense and frameshift mutations?
Silent mutations are mutated codon codes for the same amino acid. In this case there is no amino acid change Missense mutations are mutated codon codes for a different amino acid. In this type of mutation there is one amino acid change and can have a variety of effects on the organism. A nonsense mutation is when a premature stop codon is created and the result is that the polypeptide chain is shortened.This could have serious effects on the organism Deleterious. Truncated protein is typically unstable and/or nonfunctional Frameshift mutations are caused by insertions and deletions of nucleotides, which results in changes of the reading of the base sequence. This results in a shift in the entire sequence of the chain at the insertion or deletion point and can cause the chain to be too long or too short. Deleterious. Improper aa sequence inactivates protein & stop codon encountered in wrong reading frame truncates the protein
How can "silent" mutations have profound effects on protein synthesis?
Silent" mutation: does not change an amino acid, but in some cases can still have a phenotypic effect, e.g., by speeding up or slowing down protein synthesis, or by affecting splicing. "Silent" mutations can eliminate production of a functional protein •Absence of multidrug resistance correlates with either of 2 mutations in MDR1 •GGC GGT (both encode Gly412) & ATC ATT (both encode Ile1145) •Due to codon bias, GGU & AUU are rare codons relative to GGC & AUC •The ribosome "stalls" at the rare codons waiting for less abundant tRNAsto enter the A site •While translation is delayed, the part of the protein synthesized cannot fold properly & is degraded •Moral of the story: "silent" mutations are not necessarily "invisible" •There are increasing examples of proteins with identical amino acid sequences that due to differences in translation elongation rates fold improperly & are either nonfunctional or rapidly degraded
What are the consensus sequences that define splice donors, branch sites, & splice acceptors?
Splice donors, splice acceptors, and branch sites ensure the specificity of splcing Introns always have two distinct nucleotides at either end. At the 5' end the DNA nucleotides are GT [GU in the premessenger RNA (pre-mRNA)]; at the 3' end they are AG. These nucleotides are part of the splicing sites. DONOR-SPLICE: splicing site at the beginning of an intron, intron 5' left end. ACCEPTOR-SPLICE: splicing site at the end of an intron, intron 3' right end. DONOR-SPLICE ACCEPTOR-SPLICE branch point consensus sequence is yUnAy In higher eukaryotes, pre-mRNA splicing is mediated by degenerative splicing cis-elements comprised of the branch point sequence (BPS), the polypyrimidine tract (PPT), the 5′ and 3′ splice sites and exonic/intronic splicing enhancers/silencers
What are the most important features of the genetic code?
Start and Stop, stop codons can end a protein prematurely causing it to have no function
. How does CyclinD/CDK enable cells to pass the restriction point to S-phase?
Synthesizing DNA, highlighting CDK once cell has permisiionto replicate to go to cell division, expression of complex needs to increase to intiateS phase dunctionod CDK needs to decrease coming from signal outside of cell A protein known as E2F when activated will start replication, for this to happen needs to nor me inhibited by Rb CyclinD/CDK inactivates the Retinoblastoma(Rb) tumor suppressor to pass the restriction point Rbis a tumor suppressor that binds to the E2F transcription factor. E2F activates genes encoding DNA replication enzymes. Cyclin D/CDK phosphorylates & inactivates Rb. E2F now binds DNA & replication enzymes are expressed. The cell is now committed to replicate its DNA.
Why is telomerase required to maintain chromosome ends and how does it function?
Telomeres are repetitive nucleotide sequences at each end of chromosomes. Their function is to protect the ends of the chromosomes from deterioration or fusion to other chromosomes during cell division. With every cell division, telomeres shorten. Polymerase epsilon and then one unwinds it Polymerase delta finishes up relicationabt1000 nucleotides or so Ligase RNA primer removed by RNAse RNAs digest RNA w/o paying attention to the RNA sequence
What is the "Wobble Hypothesis" and why is Wobble necessary?
The Wobble Hypothesis explains why multiple codons can code for a single amino acid. ... This hence explains why more codons exist than there are specific tRNA molecules. The Wobble Hypothesis also illustrates why the only variability between many codons, that encode the same amino acid, is their 3rd base. The flexibility in base pairing b/n the 3 prime nucleotide in the codon and the 5 prime in the anti is known as the wobble The Wobble hypothesis proposes that normal base pairing can occur between nitrogen bases in positions 1 and 2 of the codon and the corresponding bases (3 and 2) in the anticodon. Actually, the base 1 in anticodon can form non-Watson-Crick base pairing with the third position of the codon. Each tRNA carries an anticodon sequence that matches to the triplet codons of the mRNA. The wobble allows for a cell to successfully synthesize proteins without needing to have all 61 codons represented in the cell's tRNA. It can also provide some protection against deleterious mutations, because a DNA mutation at the 3rd position of any codon has a good chance of not changing the amino acid inserted during translation. The wobble effect states that the third base can sometimes tolerate a mismatch because multiple codons can encode for the same amino acid
How does mRNA form a "closed loop"?
The closed loop model. By binding simultaneously to eIF4E and PABP, which in turn bind the 5′ cap and 3′ poly(A) tail, eIF4G forms a protein bridge that brings the two transcript ends together and allows regulatory information (such as deadenylation) to be transmitted from the 3′ to 5′ end of an mRNA. interaction between the 5' cap-interacting factors and the 3' poly(A)-binding protein in bringing the ends of the mRNA into close proximity and promoting both translation and stability of the mRNA, in a form known as the closed loop.
What is the relationship between exon/intron junctions and codons?
The main thing to remember is that exon and introns are features of DNA, whereas codons are features of RNA. intron-exon junction. introns are removed by the catalytic action of small nuclear riboproteins (snRNPs) which bind to special recognition sequences at the 5,(donor junction) and 3,(receptor junction) to form a complex called a spliceosome. Three common technical terms in molecular genetics, exon, intron, and codon, have specific technical definitions, but are often miss-used in hurried or short-hand presentations. The main thing to remember is that exon and introns are features of DNA, whereas codons are features of RNA. Homologous sequences in the other type of nucleic need to be called something else, otherwise there is a danger the roles of DNA and RNA in the Central Dogma ("DNA makes RNA makes Protein") will be confused. By definition, exons and introns are sequences in a protein-coding gene region of a double-stranded DNA molecule (dsDNA) that are expressed as proteins, or intervening sequences not so expressed. The exons and introns are typically shown as the single-stranded sequences of the Sense Strand of the dsDNA, written 5'-3', left to right. Transcription of the complementary Template Strand produces a heterogeneous nuclear RNA (hnRNA) that is identical (co-linear) in 5'-3' orientation and base sequences to the DNA Sense Strand, with the substitution of U for T. The RNA sequences equivalent to the DNA exons and introns are sometimes themselves referred to as "exons" and "introns," however this is technically incorrect and also confuses their functional role in transcription and translation with exons and introns as gene sequences in DNA. The RNA sequences equivalent to to DNA exons and introns can be referred to as "exon transcripts" and "intron transcripts," or "equivalents," respectively. Processing of the hnRNA to mRNA involves excision ('splicing out') of the intron transcripts and ligation of the remaining exons. Once the final mRNA is formed, translation is the process of reading (as amino acids) a series of three-base sequences called codons. Codons are read according to the Genetic Code, which is an RNA code. Because the mRNA region isequivalent to DNA exon, the same series can be identified in the Sense Strand (substituting T for U). The three-base DNA motifs are some called "codons", however this is again technically incorrect and confuses the information content of Genes with the function of RNA in the Genetic Code. The DNA equivalents to codons can be referred to as 'triplets.' In bioinformatics, the 64 triplets are sometimes presented as a "translation table" that can be used directly with the DNA Sense Strand sequence to infer the protein sequence. This is practical, except that "translation" here means 'extraction of coded information' is not the same as the molecular process of mRNA translation
. How does the spliceosome "know" which splice donor goes to which acceptor?
The spliceosome works by recognizing specific sequences that signal the border between introns and exons. Once the machine is bound to these 'splice sites' on each side of an intron, it brings the two neighboring exons close together and cuts out the intron. The two ends of the exons are then attached together.
Why is DNA ligase required for DNA replication?
•DNA polymerase I removes RNA primers & replaces them with DNA •DNA ligase joins the individual Okazaki fragments Bacterial DNA synthesis proceeds at ~1,000 nts/sec
What are Okazaki fragments?
•DNA polymerase III synthesizes DNA from 5' to 3' •Lagging strand synthesis is slower than leading strand because it requires new primers •The lagging strand "pieces" are termed Okazaki fragments & are ~ 1000 ntslong RNA primer generate an okazakifragment Leadnigstradis always faster than the lagging strand in replication
How are miRNAs produced and how do they function as silencers & destroyers of mRNA?
•This generates a 20-26 ntmiRNA*:miRNA duplex that binds to Argonauteproteins •The miRNA* strand is degraded leaving a miRNA "guide" that is complementary to its target •This forms the active RNAInduced SilencingComplex (RISC) •The RISC binds to sequences generally present in the 3'UTR of the target mRNA •mRNA fate is determined by the extent of miRNA-mRNA base pairing MicroRNAs (miRNAs) are pervasively expressed and regulate most biological functions. ... miRNAs work as small guide molecules in RNA silencing, by negatively regulating the expression of several genes both at mRNA and protein level, by degrading their mRNA target and/or by silencing translation.
Why does DNA polymerase have a "proofreading" activity?
DNA polymerases are the enzymes that build DNA in cells. During DNA replication(copying), most DNA polymerases can "check their work" with each base that they add. This process is called proofreading. ... Polymerase uses 3' to 5' exonuclease activity to remove the incorrect T from the 3' end of the new strand.
What is an origin? Why does the E.coli chromosome have a single origin, but human chromosomes require multiple origins?
DNA replication proceeds bidirectionally from fixed points called origins For DNA to start replication need to haveacessto the 2 strands, one strand must be exposed Replicatoinbubble gets biggerandreplication fork Bacteria single origin of replication Double helix is being synthesized as double helix is unwinding Problem with replication bubble? As the replication fork expands the polymerase has to restart Prokaryotic chromosomes have one origin of replication, while eukaryotic chromosomes have multiple origins. This is because eukaryotic chromosomes aremuch larger, so multiple origins are needed to replicate the entire chromosome in a short amount of time.
What is a polysome?
a cluster of ribosomes held together by a strand of messenger RNA that each ribosome is translating. The function of a polysome is to translate an mRNA into proteins.
Anticodon
a sequence of three nucleotides forming a unit of genetic code in a transfer RNA molecule, corresponding to a complementary codon in messenger RNA.
How do aminoacyl tRNA synthetases "charge" tRNAs? How accurate is amino acid charging? How are uncharged & charged tRNAs designated?
aminoacyl-tRNA connects the tRNA to the amino acid that corresponds to the anticodon Enzymes are specific, recognizing features of a certain tRNA including the anticodon Read both languages of nucleic acid and protein There si a bond b/n an amino acid and a three prime end of a corresponding tRNA A tRNA covalently bonded to its amino acid is known as a charged tRNA, this is because the bond has substantial E which is later used to drive peptide formation
What are the functions of most of the RNA synthesized in eukaryotic cells?
mRNA Function Serves as intermediary between DNA and protein; used by ribosome to direct synthesis of protein it encodes tRNA Carries the correct amino acid to the site of proteinsynthesis in the ribosome Whereas a single RNA polymerase species synthesizes all RNAs in prokaryotes, there are three different RNA polymerases in eukaryotic systems: 1. RNA polymerase I synthesizes rRNA. 2. RNA polymerase II synthesizes mRNA. In eukaryotes, the mRNA molecules always code for one protein, whereas in prokaryotes, many mRNAs code for several proteins. 3. RNA polymerase III synthesizes tRNAs as well as small nuclear and cellular RNA molecules. The eukaryotic polymerases have a more complex subunit structure than that of prokaryotic polymerases. Some of the subunits are similar to the corresponding E. coli proteins, but others are not.
How do microRNAs regulate mRNAs?
miRNAs (microRNAs) are short non-coding RNAs that regulate gene expression post-transcriptionally. They generally bind to the 3'-UTR (untranslated region) of their target mRNAs and repress protein production by destabilizing the mRNA and translational silencing.
What is the Spliceosome?
A spliceosome is a large and complex molecular machine found primarily within the nucleus of eukaryotic cells. The spliceosome is assembled from small nuclear RNAs and approximately 80 proteins. The spliceosome removes introns from a transcribed pre-mRNA, a type of primary transcript.
. Why are proto-oncogene to oncogene conversions described as "gain of function" mutations while the inactivation of tumor suppressors are termed "loss of function" mutations?
Activated oncogenes can cause those cells designated for apoptosis to survive and proliferate instead. TSG- slow down cell division, repair DNA mistakes, or tell cells when to die
How does IRE-BP mediate the iron-dependent regulation of the transferrin receptor and Ferritin?
After an mRNA is completely made, its expression can be regulated in a variety of ways: 1.RNA-binding proteins affect the translation and degradation of mRNAs Example: Iron Regulatory Protein (IRP) Iron is an essential element for the survival of living organisms because it is required for the function of many different enzymes. Transferrin protein carries iron through the bloodstream transported into the cytosol by endocytosis. Excess iron stored within a hollow, spherical protein known as ferritin. Too much iron will kill due to oxidizing, and too little leads to affected functioning, requires two proteins one is transferrin through endocytsosi, the other is ferrtitinwhich takes iron molecules and stores them (up to 4000) Excess iron can be toxic, so levels must be regulated The two mRNAs that encode ferritin and the transferrin receptor are both influenced by an RNA-binding protein known as the iron regulatory protein (IRP) binds to a regulatory element within these two mRNAs known as the iron response element (IRE) IRE in ferritin mRNA is in 5' UTR IRE in transferrin receptor mRNA is in 3' UTR Low iron: IRP binds to this IRE prevents the translationof the ferritin mRNA High iron: Iron binds to the IRP preventing it from binding to the IREàferritinmRNA is translated to make ferritin protein
How can RNA-directed exon skipping be used to treat genetic diseases?
Alternative splicnig of teh gene encoding can determine whether the antibody proteins become embedded in the membrane of the B lymphocyte that makes them or are secreted into the blood To make an antibody all exons except dor the exon #6 which has a splice donor site out of the 8 exons, encodes a hydrophobic C terminus While the first six exons including the splice donor section of the sixth, are spliced to amke a hydrophylic antibody These two mRNAs encode differnet products that are directed at differnet parts of the body human genetic diseases are caused by mutations that cause splicing defects.
Why is bidirectional DNA replication described as "semi-discontinuous?"
Bidirectional DNA replication is semi-discontinuous - One strand Leads& the other Lags •Leading strand: Same direction as replication fork: 1 Primer, continuous synthesis •Lagging strand: Opposite direction from replication fork: Multiple primers, short fragments
What is the evidence that base pairing between sequences in U1 and U2 snRNAs and complementary sequences adjacent to splice donor and branch points, respectively, are essential for pre-mRNA splicing?
Certain snRNPs base pair with the splice donor and acceptor sequences in the pRNA transcript to bring together two exons and flank an intron Pre-mRNA splicing is an essential processing step for the expression of ∼90% of protein-coding human genes and relies on conserved sequence elements at both ends of introns, termed splice sites We recently showed that restoration of base-pairing to both U1 and U6 is essential to rescue recognition of a mutant AT 5′ss that causes aberrant splicing and myotonia U1 snRNP interacts with the 5' splice site and U2 snRNP with the branch site of the pre-mRNA; both of these interactions involve Watson-Crick base pairing.
What is the basic structure and function of cyclin-dependent kinases? How are CDKs regulated?
Cyclin-dependent kinases (CDKs) are protein kinases characterized by needing a separate subunit - a cyclin - that provides domains essential for enzymatic activity. CDKs play important roles in the control of cell division and modulate transcription in response to several extra- and intracellular cues •CDKs are S/T kinases consisting of a catalytic subunit (CDK) & a regulatory Cyclin subunit •S/T kinases phosphorylate specific serines& threonineson target proteins •CDKs activate some proteins by phosphorylation & inactivate others •CDKs are activated by: •Periodic synthesis of a regulatory Cyclin subunit •CDKs are inactivated by: Periodic destruction of a regulatory Cyclin subunit Trigger cells to enter in S phase ( DNA is replicated)
How does the p53-dependent DNA damage checkpoint function?
DNA Damage Checkpoints Activate the p53 Tumor Suppressor •p53 is a transcription factor that activates expression of the CDK inhibitor, p21 •p21 inhibits CDKs and stops the cell cycle so cells do not enter S phase •p21 also inhibits CDKs in S & G2so cells do not enter Mitosis •This allows the Retinoblastoma(Rb) tumor suppressor to prevent cells from entering S phase •p53 also activates expression of DNA repair genes to fix the damage •If the damage is fixed, p53 is inactivated & cells resume the cell cycle •If the damage is notfixed, p53 activates apoptosis genes & the cells commit suicide Checkpoint failure due to mutations that inactivate p53 is a major cause of cancer
What are the main biochemical and structural determinants that distinguish RNA from DNA?
DNA contains the sugar deoxyribose, while RNA contains the sugar ribose. The only difference between ribose and deoxyribose is that ribose has one more -OH group than deoxyribose, which has -H attached to the second (2') carbon in the ring. DNA is a double-stranded molecule, while RNA is a single-stranded molecule. DNA RNA Structural Name: Deoxyribonucleic Acid Ribonucleic Acid Function: Medium of long-term storage and transmission of genetic information. Transfer the genetic code needed for the creation of proteins from the nucleus to the ribosome. This process prevents the DNA from having to leave the nucleus, so it stays safe. Without RNA, proteins could never be made. Structure: Typically a double- stranded molecule with a long chain of nucleotides. A single-stranded molecule in most of its biological roles and has a shorter chain of nucleotides. Bases/Sugars: Long polymer with a deoxyribose and phosphate backbone and four different bases: adenine, guanine, cytosine and thymine. Shorter polymer with a ribose and phosphate backbone and four different bases: adenine, guanine, cytosine, and uracil. Base Pairing: A-T (Adenine-Thymine), G-C (Guanine-Cytosine) A-U (Adenine-Uracil), G-C (Guanine-Cytosine) Stability: Deoxyribose sugar in DNA is less reactive because of C-H bonds. Stable in alkaline conditions. DNA has smaller grooves where the damaging enzyme can attach which makes it harder for the enzyme to attack DNA. Ribose sugar is more reactive because of C-OH (hydroxyl) bonds. Not stable in alkaline conditions. RNA on the other hand has larger grooves which makes it easier to be attacked by enzymes. Unique Traits: The helix geometry of DNA is of B-Form. DNA is completely protected by the body i.e. the body destroys enzymes that cleave DNA. DNA can be damaged by exposure to Ultra-violet rays. The helix geometry of RNA is of A-Form. RNA strands are continually made, broken down and reused. RNA is more resistant to damage by Ultra-violet rays.
What is the sequential order of capping, polyadenylation & pre-mRNA splicing?
5′Capping→3′ polyadenylation→splicing. Polyadenylation has been reported to occur shortly after the polymerase passes the poly(A)-addition site (Nevins and Darnell, 1978). Splicing of capproximal introns also occurs before transcription is completed
. How did the Meselson & Stahl experiment definitively demonstrate semiconservative replication?
Meselson and Stahl tested the hypothesis of DNA replication. They cultured bacteria in a 15N medium. ... This result is exactly what the semiconservativemodel predicts: half should be 15N-14N intermediate density DNA and half should be 14N-14N light density DNA. Messelson and Stahl (1958) cultured bacteria E. coli in a cultural medium containing 15N isotopes 15NH4 Cl (15N is heavy isotope of nitrogen) of nitrogen. After the replication of DNA of E. coli for many generations in 15N medium, it was found that both strands of DNA contained 15N as constituent of purines and pyrimidines. This heavy DNA molecule could be distinguished from the normal DNA by centrifugation in a cesium chloride (CsCl) density gradient. Being 15N not a radioisotopic isotope, it can be separated from 14N only based on densities. When these bacteria with incorporated 15N were placed in medium containing 14N (14NH4Cl), it was noticed newly formed DNA molecules contain one Strand heavier than the other. DNA such formed was found to be hybrid as one strand was made up of '^N (old) and another was made up of 14N (new) The various samples were separated independently on CsCl gradients to measure densities of DNA after 20 minutes (1st generation). E. coli bacterium divides in 20 minutes. During second replication (after 40 minutes) in normal 14N medium both the strands again separated (with radioactive and non-radioactive 15N). It was observed that out of total four DNA molecules formed two were completely non-radioactive and the remaining two were with one half radioactive and another half non-radioactive strand.
How can base tautomerization cause mutations?
Mutations can occur spontaneously; one such cause is tautomerization. ... The tautomer form of the basehydrogen-bonds to an incorrect base, and so the baselaid down during replication will be wrong, inducing a mutation (if this mismatch is preserved through another round of replication)..
What are the implications of 5' to 3' synthesis on replicating the antiparallel strands of double-stranded DNA?
One of the main ways DNA's antiparallel structure affects replication is in the way DNA polymerases build the new strands of DNA. ... But on the other strand (the lagging strand) the enzyme must work in the opposite direction, meaning it can only build discontinuous fragments as the double helix unwinds.
What two classes of cancer-causing mutations arise when the DNA damage checkpoint fails?
Onocogenes: Activated oncogenes can cause those cells designated for apoptosis to survive and proliferate instead. Most oncogenes began as proto-oncogenes, normal genes involved in cell growth and proliferation or inhibition of apoptosis. and tumor surpressor genes: are normal genes that slow down cell division, repair DNA mistakes, or tell cells when to die (a process known as apoptosis or programmed cell death). When tumor suppressor genes don't work properly, cells can grow out of control, which can lead to cancer.
What are the functional domains within the ribosome?
P site and E site The P-site (for peptidyl) is the second binding site for tRNA in the ribosome. ... During protein translation, the P-site holds the tRNA which is linked to the growing polypeptide chain. E site The E-site is the third and final binding site for t-RNA in the ribosome during translation, a part of protein synthesis. The "E" stands for exit, and is accompanied by the P-site (for peptidyl) which is the second binding site, and the A-site (aminoacyl), which is the first binding site.
What are the major steps involved in pre-mRNA processing in eukaryotic cells and how can these steps be regulated to determine the levels or products derived from mature mRNAs?
When an RNA transcript is first made in a eukaryotic cell, it is considered a pre-mRNA and must be processed into a messenger RNA (mRNA). A 5' cap is added to the beginning of the RNA transcript, and a 3' poly-A tail is added to the end. In splicing, some sections of the RNA transcript (introns) are removed, and the remaining sections (exons) are stuck back together. Some genes can be alternatively spliced, leading to the production of different mature mRNA molecules from the same initial transcript. Instead, it's called a pre-mRNA and has to go through some processing steps to become a mature messenger RNA (mRNA) that can be translated into a protein. These include: Addition of cap and tail molecules to the two ends of the transcript. These play a protective role, like a book's front and back covers. Removal of "junk" sequences called introns. Introns are sort of like blank or messed-up pages made during a book's printing, which have to be removed in order for the book to be readable . The 5' cap is added to the first nucleotide in the transcript during transcription. The cap is a modified guanine (G) nucleotide, and it protects the transcript from being broken down. It also helps the ribosome attach to the mRNA and start reading it to make a protein. Image of a pre-mRNA with a 5' cap and 3' poly-A tail. The 5' cap is on the 5' end of the pre-mRNA and is a modified G nucleotide. The poly-A tail is on the 3' end of the pre-mRNA and consists of a long string of A nucleotides (only a few of which are shown)
What is the role of U1 and U2 snRNAs?
Which two snRNAs bring the branchpoint sites and the 5' splice site together? U2 - recognizes the branch point site What does U1 recognize? 5' splicing site, just like U6 does What are the first three steps of RNA splicing? 1. early complex, 5' site recognized by U1 2. A complex: U2 displaces BBP by the help of U2AF (single nucleotide bulge) 3. B complex - U4,5,6 held together by base pairing bring together all the splice sites
