Module 1

DNA BLUEPRINT
- genetic information is stored in the DNA’s sequence of nitrogenous bases
- stored information can be interpreted and transmitted for cellular processes
- DNA sequences contain genes that code for proteins
- genes are transcribed into an RNA copy  DNA sequence determines RNA sequence
- mRNA is translated into a polypeptide  RNA sequence determines sequence of amino acids
- polypeptides fold into proteins that perform various cellular processes
- central dogma = process of copying genes into proteins, proposed by Crick in 1950s

DNA STRANDS
- during transcription, DNA is used to generate a complementary and antiparallel RNA molecule
- the DNA double helix is separated into two strands during transcription
- the template/antisense/minus/noncoding strand is used during transcription
- the nontemplate/sense/plus/coding strand is not used during transcription
- the nontemplate strand has the same sequence as the RNA molecule, except with T instead of U

READING THE TEMPLATE STRAND

- promoter regions indicate where transcription begins  3’ end of template strand
1. RNA polymerase binds to the promoter and separates the two strands
2. RNA polymerase moves along the template strand in a 3’ to 5’ direction
3. RNA polymerase synthesizes an RNA molecule in a 5’ to 3’ direction
4. RNA polymerase reaches the terminator sequence, disassembles, and releases the RNA transcript

CONTROL OVER TRANSCRIPTION

- for one gene, only one side of the DNA can be transcribed
- different genes may be transcribed from different sides
- which side depends on the orientation of the promoter and regulatory molecules
- most genes can only be transcribed at certain times and conditions  RNA polymerase can bind
- housekeeping genes are transcribed at all times
TRANSCRIPTION INITIATION
- the prokaryotic promoter region has consensus sequences  most common nucleotides in section
- TATAAT helps RNA polymerase recognize bind, 10bp from start site
- TTGCCA enhances rate of transcription, 35bp from start site
- RNA polymerase recruits different types of sigma factors to bind to different promoters
1. RNA polymerase binds to a sigma factor to form a holoenzyme
2. the sigma factor guides the holoenzyme to the promoters
3. the holoenzyme binds to the promoter and opens up the double helix
4. the template strand and ribonucleotides enter into the RNA polymerase through channels
5. after transcription has started, the sigma factor dissociates

TRANSCRIPTION ELONGATION: RNA POLYMERASE

- transcription occurs inside the RNA polymerase enzyme
1. the DNA double helix enters RNA polymerase through a channel
2. RNA polymerase separates the two DNA strands, forming a transcription bubble
3. ribonucleotides enter RNA polymerase and assembled into an RNA molecule on the template strand
4. the RNA molecule peels off the template strand
5. the RNA and 2 DNA strands leave the RNA polymerase through another channel
6. the two DNA strands join back together into a double helix

POLYMERIZATION
- ribonucleotides are added to the 3’ end of the RNA molecule
1. ribonucleotide triphosphate is accepted if it is complementary to the template DNA nucleotide
2. the 3’ OH forms a covalent bond with the first phosphate in the ribonucleotide triphosphate
3. the other two phosphate are released as pyrophosphate
- the formation of the phosphodiester bond is stronger and exergonic
- the release of the pyrophosphate makes polymerization irreversible

RHO INDEPENDENT TERMINATION

- rho-independent terminators have inverted repeats  sequence, reverse of sequence
1. RNA polymerase transcribes the terminator sequence
2. inverted repeats in RNA fold back into GC rich hairpin loops
3. the hairpin loops pauses RNA polymerases and separates the RNA molecule

- rho-dependent terminators use a rho factor protein

1. rho factor binds to RNA
2. rho factor uses ATP to move along RNA and unwind it from DNA
3. rho factor catches up to RNA polymerase and catalyzes dissociation of mRNA from DNA

POST TRANSCRIPTION IN PROKARYOTES

- translation occurs immediately after transcription in prokaryotes
- ribosomes may latch onto mRNA while transcription is still happening
- possible because prokaryotes don’t have a nucleus
- prokaryotic genes are differently arranged, leading to different proteins
INITIATION IN EUKARYOTES
- eukaryotes have more complex promoters, with a TATAAA sequence 30bp from the start site
- general transcription factor proteins must assemble at the promoter sequence
- a transcriptional activator protein must assemble at the enhancer sequence
- the activator recruits a mediate complex, which recruits RNA polymerase to the promoter
- RNA polymerase I transcribes rRNA, II transcribes mRNA, III transcribes tRNA

TERMINATION IN EUKARYOTES
- RNA polymerase II depends on a poly (A) mechanism of termination
- polyadenylation is coupled with termination
- termination of RNA polymerase I is similar to rho-dependent termination
- termination of RNA polymerase III is similar to rho-independent termination

POST TRANSCRIPTIONAL MODIFICATION

- 7-methylguanosine gets added to the 5’ end of mRNA through a 5’ to 5’ triphosphate linkage
- phosphatase removes one of the phosphate groups from mRNA
- guanosyl transferase attaches the 7-methylguanosine cap to the 5’ end of the mRNA
- 150-200 adenine nucleotides get added to the 3’ end of mRNA
- polyadenylation signal sequence (AATAAA) is transcribed
- poly (A) polymerase adds 150-200 adenine nucleotides  polyadenylation
- the 5’ cap is needed for ribosomes to recognize the mRNA, and protects mRNA from enzymes
- the 3’ cap stabilizes the mRNA and protects it from RNase enzymes

RNA SPLICING
- Gilbert proposed that genes contain coding (exons) and non-coding sections (introns)
- during RNA splicing, introns are removed by spliceosomes  5 small ribonucleoproteins with RNA
- introns have a donor site, branch site, and acceptor site
1. spliceosome binds to RNA at splice sites
2. a branch site nucleotide forms a phosphodiester bond with a donor site nucleotide
3. at the donor site, the intron detaches and forms a lariat intermediate loop
4. the exon at the donor site binds to the exon at the acceptor site  exons joined together
5. at the acceptor site, the intron detaches and is broken down into individual nucleotides

EXPORT OF MRNA
- after post transcriptional modification, mature mRNA is exported out of the nucleus
- mRNA moves through nuclear pore complexes
- nuclear pores = protein lined channels that go through the outer and inner nuclear membrane
- nuclear pores can transport mRNA, proteins, carbohydrates, signaling molecules
 Module 2
GAMOW’S DISCOVERY
- Gamow suggested that 3 nucleotides code for 1 amino acid
- Gamow founded the RNA Tie club with 20 members, 4 honorary members
- a 1-base code would only code for 4 amino acids
- a 2-base code would code for 4x4=16 amino acids
- a 3-base code would code for 4x4x4=64 amino acids
- therefore, it must take at least 3 bases to code for the 20 amino acids
- multiple unique triplets code for the same amino acid  redundance
- a 4 or 5 base code would be unnecessary

DECIPHERING THE CODE

- Nirenberg and Matthaei used a cell free system to decipher the first letter of the code
1. RNA, nucleotides, ribosomes, amino acids, and an energy source were placed into test tubes
2. RNA with repeated uracils coded for a polypeptide with repeated phenylalanine
3. RNA with uracils and cytosines coded for a polypeptide with serine and leucine
- confirmed that 3 nucleotides code for a specific amino acid: [UCU][CUC][UCU][CUC]
4. more RNAs were built to determine the amino acid coded by each triplet

THE GENETIC CODE

- genetic code = what amino acid each triplet codes for
- there are 64 triplets in the genetic code written in a 5’ to 3’ direction
- 61 triplets code for the 20 amino acids
- UUG, UAG, UGA stop translation, and don’t code for amino acids
- translation begins at the AUG start codon, which codes for methionine
- triplets before AUG aren’t translated
- different triplets can code for the same amino acid  redundancy
- however, each triple will only code for one amino acid  unambiguous
OPEN READING FRAMES
- the non-template strand has the same sequence as mRNA, except U is T
- scanning non-template strand for start and stop codons can be used to identify protein coding regions
- open reading frame = sequence of non-template strand from start to stop codon
- an open reading frame will code for a protein if transcribed
- however, an open reading frame may not actually code for proteins

READING FRAME
- nucleotide sequences can be read in 3 ways
- translation can begin at the 1st, 2nd, or 3rd nucleotide
- [ACU][UAC][CCG][GGA][CUA]
- A[CUU][ACC][CGG][GAC]UA
- AC[UUA][CCC][GGG][ACU]A
- reading frames lead to different amino acids and proteins
- if the side of transcription is known, there could be 6 reading frames

CRICK, BARNETT, BRENNER, WATTS-TOBIN’s EXPERIMENT

- when one nucleotide was added or removed, the code was misread
- adding a nucleotide = different codons = different amino acid sequence
- [TCA][GAC][ATA][TAC][CAT]
- [TCA][GAC][GAT][ATA][CCA]T
- adding or removing two nucleotides also led the code being removed
- added 3 nucleotides would lead to an additional amino acid, but no changes in reading frame
- removing 3 nucleotides would lead to one less amino acid, but no changes in reading frame
- therefore, 3 nucleotides code for one amino acid

RNA WORLD HYPOTHESIS

- RNA molecule was the precursor to current life
- RNA could evolved the ability to catalyze simple reactions
- RNA is needed for converting DNA to RNA
- RNA is involved in key cellular processes
- RNA world hypothesis needs further evidence
CADHERIN-CATENIN CELL ADHESION COMPLEX
- transmembrane complex that keeps epithelial cells together
- malfunction of this complex causes cancer metastasis
- metastasis = cancer spreads to vital organs, causing death

BREAST CANCER
- genetic disease that causes uncontrolled cell division, which forms a tumor and spreads
- breast cancers are classified by biomarkers
- estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor
- targeted treatments only kill tumor cells
- tamoxifen targets ER and RT breast cancer
- Herceptin targets Her(2) breast cancer
- triple negative breast cancer doesn’t have any biomarkers, and can’t be treated by targeted treatments
- triple negative breast cancer is more prevalent in young black women

KAISO
- Kaiso is a transcription factor that binds to methylated promoters and regulates 2000-3000 genes
- these genes play a role in cell proliferation, apoptosis, cell motility, and inflammation
- malfunction of kaiso = genes that control cell division and apoptosis don’t work = cancer
- patients with aggressive cancers have higher kaiso levels
- injecting kaiso depleted cells into mice inhibited tumour metastasis
- women of African ancestry had higher kaiso levels  higher triple negative breast cancer rates

GENE TO PROTEIN APPLICATION

- forest and beach mice have 1 different nucleotide in their fur colour gene
- forest mice has GCG, beach mice have ACG
- forest mice has Arg, beach mice has Cys
- as a result, forest mice have brown fur while beach mice have white fur

GENETIC CODE EXCEPTIONS

- genetic code is almost universal, meaning genes can be transferred
- in some ciliates, UAA and UAG are not stop codons, and code for glutamine
- plastids and mitochondria have their own DNA
- in yeast mitochondria, CUA codes for threonine
- in mammal mitochondria, CUA codes for leucine

TRANSCRIPTION ERRORS
- transcription errors = diabetes, dementia, Parkingson’s, aging, retinoblastoma
- retinoblastoma protein controls cell cycle progression by inhibiting transcription factors
- retinoblastoma = RB proteins doesn’t function properly, resulting in tumours on retina
- liposomes can be used to deliver chemotherapy drugs to the tumour
- chemotherapy drugs intervein during DNA replication or transcription