51.
TRANSCRIPTION
Prokaryotes – only one kind of RNA polymerase, promoter (asymmetric) – it is
conserved, sigma subunit opens helix (no ATP needed), after ~10 bases sigma falls off,
RNA synthesis speed 50 bp/sec, termination on the hairpin loop which is located before
AT region (termination signal causes RNA polymerase to fall off), RNA contains a set of
adjacent genes (polycistronic), elongation factor – DNA gyrase (uses ATP to pump
supercoils) – removes superhelical tension, transcription & translation together
Eucaryotes – RNA polymerase I, II, & III, General Transcription Factors (GTF)
assemble on TATA box, facilitate binding of RNA polymerase II, functions similar to
sigma subunit on prokaryotes, aid opening of DNA, release RNA from promoter into
elongation mode; TBP (TATA binding protein) that is part of THIID bind to TATA box,
then THIIA & THIIB add, then THIIE & THIIF bind (THIIH is on the RNA
polymerase), THIIF uses ATP to pry DNA & phosphorylate CTD; also transcription
activators need to bind GTFs & RNA polymerase), transcription activator binds to
enhancer sequence, mediator binds to RNA polymerase/GTF complex, chromatin
remodeling complex and histone acetylase add (they function in elongation & pre-mRNA
processing), RNA polymerase do not add I & II cap or tail, speed of transcription slower
= 5 bp/sec due chromatin structure, one gene per mRNA, elongation factor – DNA
topoisomerase (I & II) removes superhelical tension, RNA processing involves
capping: 7-methyl guanine added to by 5’ to 5’ linkage, 3 enzymes involved:
phosphatase removes P from 5’, guanyl transferase – adds GMP to 5’, methyl transferase
– adds meth. to G, cap binds to CBC (cap binding complex) to facilitate RNA processing
& export
splicing: 5RNAs involved (U1,U2,U4,U5,U6) that form spliceosome, >50 proteins,
needs ATP, signals to splice: 5’ splice site (by GU sequence), 3’ splice site (by AG
sequence), branch point (sequence CTRAVY), exon size ~150 bp, RNA helicase to
breaks RNA-RNA, recognize internal-external border, requires ATP (except BBP & U2),
branch point recognized by BBP & U2AF, replaced by U2 (one adenine nucleotide
becomes unpaired & attacks 5’), U1 forms base-pairs w/ 5’ splice junction, U4, U6, U5
add, U4 is rejected & falls off, U6 displaces U1 that also falls off, U5 brings 5’ & 3’,
snRNPs remain on lariat (on intron) & are recycled in nucleus by ATP hydrolysis; SR
protein – mark 5’ & 3’ splice site, 3 types: GU-AG (yeast), AT-AC (euk), GU-AT
(between 2 diff transcripts)
3’ end: CstF (AAUAA) – cleavage stimul. factor, CPSF (GU or U rich) – cleavage &
processing specif factor, bound to CTD, some CPSF bind to TFIID, cleavage of GU or U
& add of poly-A (needs ATP) – no template by poly-A polymerase (PAP) adds ~200 As
& creates a curved fiber for export
hnRNP – associated w/ introns (second abundant proteins after histones), remove hairpin
helices, mark introns for destruction, some stay bound to mRNA
Selective export – cap first, snRNP absent, proteins that mark splicing must be present,
when outside of nucleus eIF-4G & eIF-4E binds to 5’
TRANSLATION
Prokaryotes – ~31 tRNAs, smRNA 16S, lgRNA 5S & 23S , <20 aminoacyl tRNA
synthesases used, wobble base-pairing allow 20 aa among 61 codons with 31 kinds of
tRNA, speed 20 aa/sec, no cap to tell where to begin searching for the start of translation,
it contains a specific sequence (Shine Delgarno sequence – 30S/IF3 complex initiates)
before AUG of each gene on polycistronic RNA, it forms base pairs with 16S rRNA of
the small ribosomal subunit, thus ribosomes can readily assemble directly on a start
codon that lies in the interior of an mRNA molecule, fMet-tRNA initiates, elongation
factors: EF-TU, EF-G, transcription & translation simultaneous, polyribosomes to speed
up protein synthesis, release factors force addition of water to terminate (termination
entails the release of a completed polypeptide and a disassembly of the translational
machinery, three protein-release factors (RF1, RF2 and RF3) direct the termination
process in E. Coli, RF3 (GTPase) binds GTP and stimulates the binding of RF1 and RF2
to the termination codon that is positioned at the A site during the last step in elongation,
RFI recognizes stop codons UAA and UAG, RF2 recognizes sto codons UGA and UAA,
GTP expels the release factors, the remaining mRNA:ribosome complex spontaneously
dissociates to yield an mRNA plus a 70S ribosome), peptidyl transferase is catalyzed by
the RNA
Eucaryotes – ~48 tRNAs, smRNA 18S, lgRNA – 5.8S, 28S, 5S, ribosomal subunits
assembled in nucleolus, 20 aminoacyl tRNA synthesases, wobble base-pairings allow for
497 tRNAs to account for 48 anticodons, speed 2 aa/sec, monocistronic, occurs in
cytosol, in order to start translation ribosome needs to recognize the cap on mRNA,
initiation factors (eIF-2, etc.) loaded along with the initiation tRNA (only methioninecharged initiator tRNA is capable of tightly binding the small subunit) then the small
subunit with initiator tRNA scan for AUG, Met-tRNA initiates, elongation factors: EF-1,
EF-2, 5’ and 3’ ends of the mRNA interact to reinitiate translation on the same mRNA
molecule, leaky scanning of AUG – depends on sequences next AUG (it can cause a
different N terminus), release factors force addition of water to terminate (most
eukaryotic systems appear to contain only a single release factor (eRF) which catalyzes
GTP hydrolysis and recognizes all of the termination codons), peptidyl transferase is
catalyzed by the RNA
TRANSLATION PROCESS OVERVIEW
Aminoacyl tRNA synthetase – adds aa to tRNA, requires ATP (AMP high energy bond
– adenylated aa) & ATP is used to add to tRNA, correct aa = highest affinity, 2nd pocket
compares dimensions & edits (similar in function to DNA polymerase), two ATPs total
used for charging tRNA.
1) Ribosomal assembly – tRNA-Met & eIF-2 come to the P site of sm rRNA, sm rRNA
recognize cap & scans for AUG (ATP used), eIF-2 hydrolyze GTP & leave, lg rRNA
binds.
2) Elongation – aa on tRNA w/ EF-Tu bound to it – codon/antic base pairing hydrolyze
GTP (faster hydrolysis w/ correct aa) & puts aa in A site forming base pairs with the
codon in mRNA positioned there, so that the P-site and the A-site contain adjacent bound
tRNAs. Then the carboxyl end of the polypeptide chain is released from the tRNA at the
P-site and joined to the free amino group of the amino acid linked to the tRNA at the Asite, forming a new peptide bond (formed by peptidyl transferase). A/P & P/E hybrid
state & hydrolysis of GTP put in E & P & translocation occurs. Conformational changes
move mRNA exactly three nucleotides through the ribosome and resets the ribosome for
the next aa.
3) Termination – stop codon not recognized by tRNA – recognized by release factor
protein causing peptidyl transferase to add water, C terminal frees & ribosome
disassemble.
52.
DNA packaging – 5 levels:
1) DNA helix – first order of chromatin packaging, 2 nm
2) beads (3X compaction) – 11 nm, histone (2A, 2B, 3, 4) octamers producing
nucleosomes that bind to AT regions of DNA (due their positive charges in their
active binding sites). The string is DNA and each bead is a “nucleosome core
particle” that consists of DNA wound around a protein core formed from histones.
Linker DNA is considered the DNA between each nucleosome and nucleosome
cores are the histone complex and the wrapped DNA. The nucleosome has 146
nucleotides of DNA wrapped around it reducing the size of DNA to 1/3 of its
initial length.
3) 30 nm (100X) – facilitated by H1 tails that pull nucleosomes together. Histone
proteins bind to each nucleosome, contacting both DNA and protein, and
changing the path of the DNA as it exits from nucleosome.
4) loops and coil B (1000) – 300 nm, protruding from linear axis, active genes in
loops (transcribed along scaffold), converted to loop by modifying enzymes,
remodel. complexes (observed in amphibian oocytes in Lampbrush chromosome
– meiotically paired chromosomes, and banding pattern observed in Polytene
chromosomes that contain 1024 identical DNA strands, alterating bands (95%),
interbands (5%), dynamic, banding pattern the same but bands can expand &
shrink)
5) higher condensation (10000X) – 700 nm, ten times smaller than in interphase,
protects fragile DNA & this structure makes it easy to separate, condensing
proteins – use ATP to coil (GC regions less condensed & more actively
transcribed - you can see AT on Polytene chromosome, Geisma stain – GT
regions visible)
All these help with compaction of a long DNA into a small condensed structure
Chromosome is composed of:
Heterochromatin – highly packaged & organized, dynamic (genes can be reactivated
due modifications of histones), 10%, resistant to gene expression, includes telomeres,
centromere (may protects from transposable elements), has proteins associated with it
Euchromatin – less condensed, 90%, transcribable
53.
DNA Replication:
DNA polymerase – good nucleot greater affinity, has a proofread capability, synthesizes
DNA strand in 5’ to 3’ direction
DNA primase – use rNTPs to make primer (spaced 100-200 bp due nucleosomes), RNA
primers removed by RNaseH & replaced by DNA polymerase & sealed by DNA ligase
DNA ligase – joins DNA fragments (5’ end adds P (from ATP), 3’ OH makes bond &
one P falls off)
DNA helicase – uses ATP when bound to ssDNA to unwind dsDNA, moves 1000
bp/sec, two needed per each origin of replication (move opposite directions), loosen
histones not removed, unidirectional, SSB aid by destabilizing unwound ss preventing
hairpin helices but don’t cover bases
DNA topoisomerase – removes torsion strain, uses ATP, I = single stranded break, II =
double, breaks phosphodiester bonds, has tyrosine in active site – energy stored is used to
seal the DNA strands
Primosome – primase & helicase (in prokaryotes), synthesizes primers (eukaryotic DNA
pol alpha w/ DNA primase puts down primer then delta comes in)
Proteins involved in DNA replication:
Clamp protein – forms ring (sliding clamp) release DNA polymerase when runs into
dsDNA, assembly requires ATP hydrolysis by a special protein complex called clamp
loader (dissociates when DNA polymerase attaches to leading strand but stays in on
lagging)
dsDNA is coiled (not shown here)