DNA Structure, Replication, Transcription, and Translation
DNA Structure and Replication
DNA:
Constitutes the genetic material.
DNA structure:
Double helix formed of antiparallel strands.
Diameter is 20 Angstroms (20 * 10^-10 meters).
One helical turn is 34 Angstroms.
3. 4 Angstroms between base pairs.
Replication:
Semi-conservative: Each copy has one parental and one new strand.
Bidirectional.
Methods using simplified DNA replication steps: PCR and DNA sequencing.
Griffith’s experiment (1928):
Established there is a transformation factor that transforms bacteria from a non-lethal strain to a lethal, virulent strain.
Two types of Pneumococcus:
Virulent type: S type; if injected, kills the mouse.
Non-virulent type: R type; bacteria are killed by the mouse’s immune system.
Heat-killed S strain: bacteria are inactivated, and mice survive.
Mixing heat-killed S type and living R type can recreate a lethal type of bacteria.
Transformation factor transforms R type into S type from heat-killed S type.
The R type in the blood acquires a factor from the S type, transforming them into S and killing the mice.
Avery, McLeod, and McCarty (1944):
Transformation is disrupted by DNase.
DNA is the transforming factor.
Did in vitro experiments by plating the cells and looking at the morphology of the colonies, away from mice.
Plated bacteria on agar plates, took extracts from heat-killed S bacteria and mixed with live R type.
Treated the extracts with different conditions, destroying lipids, proteins, RNA, and DNA.
When DNase was added, DNA was destroyed, and no bacteria were recovered.
When the other components were destroyed, S bacteria were recovered.
Hershey-Chase experiment:
Confirmed that DNA is heritable genetic information.
Bacteriophage T4 DNA is the hereditary molecule during infection.
Grew bacteriophage in radioactive phosphate; phosphate/P-32 incorporated into DNA
Bacteriophage used to infect E. coli, and DNA finds itself into the cell and then can make the proteins required for further viral assembly
Radioactive sulfur incorporates into proteins; found cells devoid of S-35, which was found itself into the empty phage particles.
DNA has phosphate, and protein has sulfur.
DNA structure:
Chargaff’s findings: DNA is composed of 4 nucleotides.
Chargaff’s rule: In DNA, %A = %T and %G = %C. The ratio G/C = 1 and A/T = 1.
The percentage of nucleotides can vary between organisms.
DNA is a right-handed helix.
Strands are complementary and anti-parallel.
G pairs with C with 3 hydrogen bonds; A pairs with T with 2 hydrogen bonds.
5’ end is a phosphate end; 3’ end is a hydroxy end.
Rosalind Franklin:
DNA is a double helix, which is why the X formed in the photo.
Photograph 51.
Nucleotides:
The building blocks consist of sugar, a nitrogenous base, and a phosphate group.
Sugar: Pentose called deoxyribose; deoxy because of the C2 position and doesn’t have a hydroxyl group.
1’ links to the nitrogenous base.
3’ has a hydroxyl group.
5’ links to phosphate groups: 1 (mono), 2 (di), 3 (tri) phosphate groups.
Phosphodiester bonds: 5’ to 3’ linkage that connects nitrogenous base and phosphate group.
Nitrogenous base: Purines and pyrimidines.
Purines: Two rings and are A and G.
Pyrimidines: Single 6-atom ring; T and C.
Base Pairing:
G pairs with C through 3 hydrogen bonds; non-covalent; low energy bond.
A pairs with T through 2 hydrogen bonds.
All nucleic acid strands are antiparallel: DNA, RNA duplex, and DNA/RNA hybrids.
Purines pair with pyrimidines.
DNA structure (continued):
During replication, there is a triphosphate.
The alpha phosphate covalently links to the 5’ C.
Two phosphates/pyrophosphates are eliminated/hydrolyzed and provide energy.
The chain is synthesized by the addition of a nucleotide through the formation of a phosphodiester bond and the elimination of PPi (pyrophosphate).
Deoxyribonucleotides:
3 forms of DNA:
B-form: Predominant; the hydrated form in cells.
A-form: Dehydrated.
Z-form: Left-handed and only present in a small number of locations.
DNA replication:
3 possible mechanisms: Conservative, semiconservative, and dispersive.
Meselson & Stahl distinguished that it was semiconservative.
Dispersive: each strand has new and parental DNA.
Conservative: one DNA helix is fully parental and one is fully new.
Meselson-Stahl Experiment Setup:
Grow E. coli cells on heavy nitrogen (15N).
Nitrogen isotopes incorporate into DNA during replication.
Overtime, all chromosomal DNA is composed of heavy DNA.
Transfer to light nitrogen (14N) for 1 generation.
Extract chromosomal DNA and run DNA on a Cesium Chloride gradient.
DNA molecules run in the gradient according to density: light, hybrid, and heavy.
Cycle 1: Excludes conservative replication because all DNA molecules were intermediate between light and heavy; otherwise, there would be one heavy and one light.
Cycle 2: Excludes dispersive replication (otherwise would get an intermediate band); got half light and half hybrid.
Replication proceeds bidirectionally from an origin of replication.
The two directions of progression are sustained by replication forks, moving in opposite ways.
This was first demonstrated by pulse-chase experiments.
E. coli origin (ORI) found using electron microscopy:
In the image, you see initial bubble formation/unwinding of strands.
Used radioactive nucleotides; radioactivity incorporated into DNA and detected on photographic film.
At the replication fork, there is radioactivity; progression of forks can be seen with radioactivity.
ORI in E. coli:
Single ORI.
Chromosome is circular; the total length of DNA is 4.6 million nucleotides.
OriC is where the origin of replication is in E. coli.
The forks meet at the other end.
Topoisomerases intervene, cut the DNA strands, allow them to rebind, and religate to form two molecules of DNA.
Each new bacteria has an old strand and a new strand.
Huberman and Riggs experiment:
Feed hamster cells with radioactive dTTP for 1 hour (pulse).
Pulse period: The period where radioactivity can incorporate into DNA during synthesis.
Add an excess of non-radioactive dTTP (chase) to continue replication but stop the incorporation of radioactivity in DNA.
Extract strings of DNA and lay them on a solid surface to expose them to film.
On top, you see a zone that is clear and a zone that has radioactivity, then another weakly labeled zone, etc.; shows the progression of replication forks away from each other.
Eukaryotic Replication:
Eukaryotic genomes are replicated with many origins of replication.
The replication forks meet, and newly made strands are processed and ligated.
Each origin gives rise to two forks.
The zone of replication is about 30k-50k.
Each new strand starts with an RNA primer that is later excised and degraded; newly made strands are processed so DNA nucleotides replace the RNA nucleotides.
Once DNA replaces RNA, the DNA strands are ligated together.
Mechanism of replication:
Replication always occurs in the 5’ to 3’ direction; extension of the 3’ end; new nucleotides are added one by one to the 3’ OH of the deoxyribose.
Replication requires a primer, itself presenting a free 3’ OH.
For chromosomal DNA replication, there is an RNA primer; for PCR or DNA sequencing, a synthetic DNA primer is used.
Major Enzymes:
Topoisomerases: Associate with the double helix and cleave the two strands to let DNA unwind; later religate; essential to resolve torsion produced by the progression of replication forks; do so ahead of helicase.
Helicase: Unwinds the double helix; called DnaB in E. Coli; the first activity recruited at the replication fork in E. Coli; breaks hydrogen bonds; two helicases at each bubble, hydrolyzes ATP to unwind the strands.
Single-stranded binding protein: Prevents reannealing of separated strands.
Primase: Synthesizes RNA primers; recruited right after helicase and SSB; primase synthesizes dinovo, but DNA poly doesn’t
DNA polymerases can only elongate a pre-existing 3’ end.
DNA Poly III: Synthesizes the DNA; adds DNA nucleotides to 3’ ends.
DNA Poly I: Removes and replaces RNA primer with DNA; has a role in the removal and replacement of RNA primer nucleotides; does this in several cycles of catalysis to replace RNA nucleotides one by one
Ligase: Joins DNA segments; catalyzes a phosphodiester bond between 5’ and 3’ ends.
Initiation, elongation, and termination.
Termination done by DNA Pol I and ligase.
Leading and Lagging Strand:
The parental strands have opposite polarities.
One parental strand can be replicated in the same direction as the fork’s progression.
Leading strand synthesis: Continuous replication by extending the 3’ end.
The other strand is replicated discontinuously; the lagging strand.
Leading and lagging strand synthesis has to happen in the 5’ to 3’ direction.
For the lagging strand, primase has to generate several primers along the strand.
Okazaki fragments are the small fragments in lagging strand synthesis.
Each parental strand is replicated by leading and lagging strand synthesis, depending on which fork you are looking at.
One enzymatic complex replicates each strand.
The final products are hybrid duplexes.
The replisome connects DNA replication.
E. coli OriC
OriC is a specific DNA sequence recognized by proteins.
There is a protein called DNAA that recognizes some of this sequence.
Have 3 copies of a 13 bp sequence and 4 copies of a 9 bp sequence; a tandem repeat array that makes up OriC.
Bacterial ORI:
Consensus sequences: A set of nucleotides that can be fairly short or long and appear at the same position at a regulatory sequence (ex: promoters) in different contexts.
Can have the same sequence in different bacteria.
The bacteria here have the same or very similar consensus sequences.
Eukaryotic Consensus Sequences:
Consensus sequences work in unicellular eukaryotes.
ARS1: Yeast origin.
There are 100s of such origins on each chromosome.
Origins in higher eukaryotes like mammals aren’t well-defined in sequence.
Proteins used are conserved, mainly among eukaryotes, though.
Mechanism of recognition at the origin C:
DnaA is an initiator protein that recognizes 9 tandem repeats.
DnaA binds to the 9-mer region, forcing unwinding of the 13-mer region to form an open complex.
DnaC delivers DnaB protein (helicase in E. Coli) to the open complex to initiate helicase activity.
The mer sequences are AT-rich so they're easier to unwind.
Additional proteins join to form the primosome.
DnaA and DnaB are 6-unit complexes; DnaC is a 1-unit complex.
Removal of RNA primers:
Poly I has the role of removal and replacement of RNA primer nucleotides.
DNA Poly I does this in several cycles of catalysis to replace RNA nucleotides one by one.
DNA ligase III ligates Okazaki fragments.
Selected Bacterial and Eukaryotic Polymerases:
DnaG - RNA primer synthesis; primase in bacteria.
DNA poly I - RNA primer removal, proofreading, mutation repair in bacteria.
DNA poly III - DNA replication, proofreading.
DNA poly alpha - Primer synthesis and lagging strand synthesis in eukaryotes.
DNA poly beta - Lagging strand synthesis, proofreading, DNA mutation repair.
DNA poly epsilon - Leading strand synthesis, proofreading, DNA mutation repair.
Sliding clamp:
Processivity factor.
Processivity: The same enzyme molecule or enzymatic complex performs many rounds of catalysis without disjoining from the substrate.
Poly III stays bound to DNA, and the same enzyme can undergo thousands of rounds of catalysis without leaving DNA.
Processivity makes replication faster and more efficient.
The clamp assembles around DNA and makes contact with Poly III, allowing retention of the enzyme on DNA at each round of catalysis.
Proofreading:
DNA polymerases have proofreading activity.
DNA Poly II is an exonuclease site.
Occasionally, there is a mismatched base pair.
The daughter strand rotates out of the polymerase site and into the exonuclease site; this one is a 3’ exonuclease site, meaning it can cleave a nucleotide at the 3’ position.
The daughter strand then re-rotates and resumes DNA synthesis.
DNA poly III has a polymerase domain and an exonuclease domain, giving rise to different functions.
Topoisomerase:
Supercoiling occurs during the replication of a circular molecule.
Topoisomerase cleaves DNA at that position and allows relaxation of DNA and then rejoins the strands.
Especially important on circular molecules like the E. coli chromosome; also present in eukaryotic replication.
End Replication Problem:
Problem on linear molecules.
The end replication problem affects both strands at both extremities at their respective 3’ ends.
On the lagging strand, there is a single-stranded overhang left by RNA primer removal at the telomere.
The terminal 3’ end of the linear DNA molecule can’t fully be replicated due to the need for a primer to initiate replication.
Telomerase:
The holoenzyme telomerase, a reverse transcriptase, bypasses the end replication problem by adding telomeric repeats to chromosome ends.
The addition of repeats by telomerase at the 3’ end provides additional space for laying down an RNA primer and prevents telomere shortening.
2-30 kb double-stranded TTAGGG repeats; in humans, 6-10 kb.
This repeat is found in humans and all eukaryotes.
500-300 nt single-stranded overhang.
Folds in the t-loop structure through strand invasion.
The complex that associates with telomeres is called Shelterin.
Shelterin protects telomeres against degradation.
Excessive telomere shortening leads to permanent cell cycle arrest or cell death.
PCR:
3-50 cycles of denaturation, annealing, and extension.
Denaturation at 95 degrees Celsius; annealing at 45-68 degrees Celsius; extension in the presence of dNTPs.
72 degrees Celsius is the ideal temperature for Taq Polymerase, a heat-resistant enzyme.
DNA Sequencing:
Use of dideoxynucleotide NTPs.
ddNTPs are 2’,3’ dideoxy nucleotides that can’t support chain elongation.
ddNTPs can be used by DNA poly but can’t be extended due to the 3’ deoxy group.
Chain termination by ddNTPs at each position they are incorporated.
Sanger Method
DNA-sequencing gel: can run fragments and give you the sequence of the template used in the reaction.
Reading the sequence away from primer site, bottom to top.
Next Gen Sequencing:
NGS or Illumina sequencing: all in one tube, using fluorescently labeled dNTPs.
At each round, the newly incorporated nucleotide is identified by its emission profile.
Molecular Biology of Transcription and RNA Processing
RNA:
RNAs are transcribed from DNA as mRNA, rRNA, tRNA, snRNA, miRNA.
Transcription is a 4-step process: promoter recognition, initiation, elongation, termination.
Poly I transcribes rRNA, Poly II transcribes mRNA, Poly III transcribes tRNA and miRNA.
mRNA processing is essential for gene expression: capping, splicing, polyadenylation (poly A addition).
RNA Structure:
Ribonucleotides: The sugar is a ribose with a 2’ OH group.
RNA has 4 nucleotides: A, U, G, and C.
RNA Synthesis:
5’ to 3’.
NTPs are hydrolyzed, and there is a pyrophosphate release (PPi).
The chain grows 5’ to 3’; bonds between nucleotides are phosphodiester bonds between 5’ phosphate and 3’ hydroxyl.
Types of RNA:
mRNA - Protein-coding; used by ribosomes for translation and polypeptide synthesis.
rRNA - Long transcript; not translated; interacts with ribosomal proteins and forms the ribosome; they direct the actual growth of the polypeptide based on coding sequence.
tRNA - Amino acid carriers; contain an anticodon that is a reverse complement to each codon present on mRNA.
snRNA - Important for splicing.
siRNA - Eukaryotic gene expression.
Telomerase RNA - Type of snRNA; required to elongate ends of chromosomes.
Transcription:
promoter region recruits the RNA polymerase that will perform transcription; upstream of sequence being transcribed/coding region
Poly II transcribes mRNA.
The presence of introns interrupts the coding sequence in mRNA; later removed in RNA processing.
Transcription for protein coding genes is dependent on RNA Poly II.
RNA Polymerase:
Elongation in the 5’ to 3’ direction.
RNA produced is complementary to the DNA template.
U is a complementary base to A in the DNA.
RNA is produced antiparallel to the DNA template.
The DNA strand being transcribed is the template.
The template strand is reverse complement to the RNA sequence.
There is a non-template/coding strand that isn’t being transcribed; has the same sequence as RNA except RNA has U’s.
Depending on the transcription unit of the genome, a specific DNA strand can be the coding or template strand for different genes.
Direction of Transcription:
More than one promoter can be used.
Many isoforms are generated by alternative splicing.
A segment of chromosome 7 with 4 genes; the direction of transcription can go either way, depending on the strand being produced.
Promoter:
Here is a Poly II promoter for mRNA/protein-coding genes
Eukaryotic promoter elements: the basal promoter/TATA box around position -25, the CAAT box, and the GC-rich box.
Note: +1 corresponds to the transcription start: the first nucleotide to be transcribed.
The bottom strand is being transcribed from left to right.
Variability in the structure of eukaryotic Poly II promoters
TATA boxes are present pretty much all the time.
CAAT and GC boxes may or may not be there.
Quantities may vary too.
Band Shift Assay:
Used to determine the DNA segment that contains a binding site/promoter consensus sequence for the protein.
The DNA is end-labeled with radioactive phosphate and mixed with protein to allow for binding and run on a gel.
The protein-DNA complex runs as a higher molecular weight product and has slower migration.
Footprint/Protection Assay:
To determine the area covered by RNA poly on the DNA, including the promoter, DNA is end-labeled with radioactive phosphate mixed with protein to allow binding to occur.
Then digested with DNase 1 which cleaves DNA as long as it has access to it; if protein is bound, it won’t be able to cleave it.
There is an area on the DNA which is protected from digestion.
On the gel, DNA is clipped at almost every nucleotide.
On the left, if there is Poly, there is no cleavage due to its protection.
Initiation of Transcription:
TATA is recognized by TFIID.
TFIID contains a subunit called TBP (TATA-binding); TBP makes contact with TATA.
Also have TAF which complexes them together.
There are also TFIIA and TFIIB, which help recruit RNA poly.
RNA polymerase is recruited at the right position to initiate transcription at the +1 side.
When TFIIE and TFIIH are there, a preinitiation complex is formed.
Once RNA poly II comes in, there is a subunit of the complex called TFIIF, which has helicase activity and forms a bubble at that position.
TFIIF allows RNA Poly II to start transcription by creating a bubble.
Transcription factors are released, and RNA Poly II synthesizes mRNA in the 5’ to 3’ direction.
Mutational Analysis of Beta-Globin Gene Promoter:
+1 A is the transcription start.
Analysis of base substitution in the genes’ regulatory regions.
Most mutations have no impact, some reduce transcription, and some increase it.
When you change the TATA box, CAAT box, or GC-rich box, transcription is reduced.
2 Gs in the CAAT box are inhibitory to transcription.
A balance of regulation must be achieved.
Other Elements: Enhancers
Elements aren’t part of the promoter; they can be very far away.
Enhancers: Elements that bind transcription factors that are called activator proteins.
Activator proteins can recruit coactivator proteins/mediators.
They make contact with the pre-initiation complex.
They help in the assembly and stability of the pre-initiation complex.
These elements can increase transcription 100X fold or 1000X fold.
Not part of the basal regulatory region.
Can be present upstream or downstream of the gene.
Not found in all promoters.
The effect of these elements is possible because of DNA looping.
An enhancer needs a promoter to have an effect on transcription; it can affect multiple basal promoters.
Transcription Initiation by Poly I:
Have an upstream control element at around -100 that increases transcription efficiency.
Core element: Initiates transcription; -45 to +20.
UBF1 and SL1 bind to upstream control and core elements; RNA Poly I is recruited to the core element to initiate transcription.
Poly III:
An internal promoter contains box A and box C.
Have an ICR (internal control region); part of the unit being transcribed.
Box A recruits TFIIIC, and box C recruits TFIIIA.
TFIIIB binds to the two, and RNA poly III binds to TFs and is positioned at +1.
Transcription by Poly III at tRNA genes
Archaeal:
Archaeal promoter consensus sequence.
Looks more related to prokaryotic consensus sequences.
RNA Processing:
Transcription + mRNA processing occurs in the nucleus.
Translation (protein synthesis) occurs in the cytoplasm.
After mRNA processing, the mRNA leaves the nucleus during the regulation of the mRNA process.
A 5’ cap and poly A tail are added; introns are removed.
Capping:
Guanylyl transferase catalyzes this reaction.
Guanine monophosphate is joined to the 5’ mRNA end by a 5’ to 5’ triphosphate linkage.
Additional methylation of nucleotides may occur; this is catalyzed by phosphate hydrolysis.
Capping is important for stability, export, splicing, translation.
Alpha, beta, and gamma differentiate the phosphates bound; alpha is closest, and gamma is most external.
Alpha and beta phosphates come from the transcript itself, and the gamma is the phosphate from the guanine monophosphate.
Polyadenylation:
A post-transcriptional event.
Cleavage of mRNA occurs 15-30 nucleotides following the polyA site; the cleavage happens when mRNA is still being transcribed.
Cleavage is downstream of the coding sequence.
PAP = Poly A polymerase adds 20-200 A’s to the 3’ end.
Important for stability, export, and translation.
Signals are present that are functional on the transcript.
The Polyadenylation signal sequence: AAUAAA.
There is also a U-rich region further downstream; 50-100 nucleotides; important for the overall reaction.
Between the two signals is the cleavage site.
CPSF - Cleavage polyadenylation specificity factor.
CStf - Cleavage stimulation factor.
CPSF and CStf and CFI and CFII factors form on the 3’ region of the transcript as it's being made.
CFI and CFII form the cleavage.
PAP extends and has an associated factor later on called PABII.
Transcription Termination:
Happens after poly a adenylation.
The role of the torpedo RNase in eukaryotic transcription termination; degrades the RNA and meets with RNA poly II, leading to dissociation.
Torpedo is a 5’ to 3’ nuclease attacking the exposed (uncapped) 5’ following 3’ cleavage by CFI/CFII.
Mechanisms of transcription termination in prokaryotic transcription are very different.
The poly A tail really helps end transcription.
Splicing:
Removal of introns.
Spliceosome - Used for eukaryotic nuclear genes, targeting the pre-mRNA introns; made up of ribonucleoproteins
rRNA and tRNA introns are processed enzymatically.
Introns:
Non-coding sequences that are part of DNA but interrupt the coding sequence.
The coding sequence is a continuous set of 3-nucleotide codons that are translated by ribosomes and form proteins.
Intron Proof:
EM evidence for introns.
Detection of R-loops: Hybridization of genomic DNA with corresponding mRNA.
There are sequences in the DNA that don’t hybridize and loop out: the introns.
Introns were discovered, though, by virologists; extracted RNA after infection of cells and found when hybridized viral RNA with viral DNA had the same result.
Splicing Reaction:
Intron removal by the spliceosome.
The splice donor site on the left and the splice acceptor site on right are important signals.
The 5’ splice donor site on the left is a very weak consensus and is small and can be present in many places; GUAGU is important; GU is most important.
A stretch of pyrimidines and CAG is the 3’ splice site/acceptor site on the right side.
Branch site: 20-40 nucleotides upstream of the 3’ splice site: pyrimidine-rich with a branch point adenine/essential adenine for reaction.
A key intermediate is called the lariat constructed by cleaving just upstream of the G in the 5’ splice site and forming a covalent bond between the G and the branch point adenine.
There is a phosphodiester bond between the 5’ guanine and the branch point on 2’ adenine.
After the formation of the lariat, there is cleavage after the 3’ splice site G and then ligation of exons 1 and 2 and the degradation of the looped molecule that has the 5’ G-2’ A.
5’ intron cleavage: The spliceosome contains subunits U1 and U2, which contain RNA and protein; RNAs can hybridize and recognize the site; U1 binds to the 5’ splice site and U2 binds to the branch site.
Lariat formation, then 3’ intron cleavage, then exon-exon ligation and lariat release
Coupling Transcription and mRNA Processing in Nucleus:
RNA poly II has a huge domain called CTD or a C-terminal domain.
The CTD recruits the polyadenylation factors, including PAP; it recruits the torpedo and the capping enzyme.
The phosphorylation of CTD is associated with transcription and the clearance of RNA poly II from the promoter.
Then recruit the spliceosome as signals emerge from mRNA.
Alternative Splicing:
Rarely have one 5’ splice site and one 3’ splice site that are matched and brought together.
In many cases, have alternative splicing.
The 5’ splice site for example, downstream of exon 3 can be paired with a 3’ splice site on exon 4 or can be paired with a 3’ splice site on exon 5.
If have 5’ from exon 3 and 3’ from exon 4, mature mRNA is formed.
If not have the 3’ splice site from exon 5 and gives a totally different structure; CGRP mature mRNA is formed.
Calcitonin and calcitonin gene-related peptide (CGRP) is an example of alternative splicing.
In this example, the acceptor splice site is regulated, but donor splice site choice can also vary.
Rat Alpha-Tropomyosin Gene:
A complex set of promoters, poly A sites, and alternative splicing events.
A single gene can produce many isoforms, which may differ from each other in their coding sequence due to alternative splicing.
Self-Splicing Introns:
Some RNAs are produced and can spontaneously undergo the splicing reaction and elimination of an intron through nucleophilic attack of G to a phosphodiester bond cleaving 5’ of the intron.
Release of the 3’ end will lead to an attack on the other end of the intron, eliminating the intron.
Don’t need enzymes or a spliceosome.
Processing of pre-RNA transcripts:
In humans, initially, a 45S pre-RNA transcript is produced (S = biophysical parameter of sedimentation, Svedberg unit); it is then cleaved to produce 28S, 18S, and 5.8S rRNAs.
These rRNAs are part of the large and small subunits in ribosomes.
In prokaryotes, you get a large 30S pre-RNA that produces 16S, 23S, and 5S.
There are also tRNA genes interspersed that are processed and will eventually form tRNA and are part of the rRNA transcription unit.
In humans, tRNAs are transcribed separately.
tRNA:
Forms a T-shaped structure.
tRNA transcript has modifications: processing of the 5’ and 3’ ends.
There’s elimination of sequences, the addition of CCA, and modifications of some nucleotides by methylation.
Most tRNAs have different anticodons that correspond to an amino acid.
tRNA synthetase recognizes the anticodon and promotes the binding of an amino acid on the 3’ OH of the ribose to the 3’ CCA of the tRNA.
The anticodon is reverse complement of one codon in the mRNA coding sequence.
The amino acid itself is bound by a carboxyl hydroxy bond to the 3’ of the adenine present at the 3’ end of tRNA.
RNA editing:
The addition of U nucleotides not encoded in DNA.
Requires a guide RNA that determines the sites of U addition.
The edited RNA has U’s at different portions.
Post-transcriptionally done.
Sex determination in flies:
Depends on X chromosomes.
In humans, the presence of Y means male, and if Y isn’t there, it's female.
In flies, XX is female because of the number of X chromosomes compared to the number of autosomes; this ratio is 1 in females.
In males, the ratio is 1:2 or 0.5.
In vitro example of regulated splicing determines sex in Drosophila.
SisA and SisB are produced by the X chromosome and interact with a protein called Deadpan.
If you have an excess of SisA and SisB, you can overcome deadpan to have the production of the SxI protein.
SxI (sex lethal) is a splicing factor produced in female embryos only.
The production of SxI depends on the ratio of number of X chromosomes/autosomes.
SxI leads to the Tra gene, which leads to the production of the Tra protein such that exon 1 and 3 are ligated together.
Tra itself is an alternative splicing factor, which is involved in the alternative splicing of Dsx in females.
The female-specific Dsx activates female genes and represses male genes.
The male-specific Dsx protein represses female genes.
Female and male Dsx isoforms differentially regulate male and female specific gene expression.
Molecular Biology of Translation
Translation:
The process