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The genetic code Transcription

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Correlation of polarities in DNA, mRNA and polypeptide


GENE Non-template strand

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Moving from 5 to 3 end of an mRNA, each successive codon is sequentially interpreted into an amino acid, starting with the N-terminus and ending with the C-terminus

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Nonsense codons
Three different triplets, UAA, UAG and UGA, do not correspond to any of the amino acids. When these codons appear in frame, translation stops, and the polypeptide chain is terminated. They are also known as stop codons.

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The genetic code is almost universal


Exceptions: in some species of single-celled eukaryotic protozoans (ciliates) the codons UAA and UAG specify glutamine differences in mitochondria i.e. those of yeast specify threonine with CUA instead of leucine

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5 in mRNA

3 in mRNA Code consists of triplet codons codons-each specifies an amino acid Codons are nonoverlapping Code includes 3 stop codons, do not code for any amino acids nonsense codons, must be in frame with start codon Code is degenerate -one codon specifies more than one amino acid

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Transcription

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Central dogma
ZOOM IN tRNA transcription DNA rRNA snRNA translation mRNA POLYPEPTIDE

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Transcription key steps


DNA

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Initiation Elongation Termination

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Transcription key steps


DNA

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Initiation Elongation Termination

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Transcription key steps


DNA

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Initiation Elongation Termination


+
RNA DNA

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Promoters
Promoters are sequences in the DNA just upstream of transcripts that define the sites of initiation. 5 3
Promoter

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The role of the promoter is to attract RNA polymerase to the correct start site so transcription can be initiated.

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Transcription
Transfer of information from dsDNA to a ssRNA

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Polymerization of ribonucleotides guided by complementary base pairing produces an RNA transcript of a gene no primer is necessary One strand of DNA is used (template-strand) (template Nucleotides are added in the 5-to-3 direction 5-to Uracil is incorporated in place of Thymine in RNA

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Transcription Process: Overview


RNA-like strand

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RNA polymerase (RNAP)- enzyme which catalyzes (RNAP)transcription unwinding of DNA (transcription bubble) near gene before transcription can begin - prokaryotes RNAP does this

Like Replication initiation elongation 3 stages:

termination

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Transcription: organization of a gene


Genes are flanked by regions called Promoters

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Promoters DNA sequences near the beginning of genes that signal RNA polymerase where to begin transcription Terminators Sequences in the RNA product that tells RNA polymerase where to stop (encoded by the DNA)

+1 site

Negative numbers

Positive numbers

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The steps in transcription: Initiation Prokaryotes


RNAP: large protein holoenzyme exits as a holoenzyme core enzyme (multiple subunits) factor - initiation only

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core

factor essential for promoter recognition increases affinity of RNA core for promoter region allows RNAP to bind tightly to promoter region aligns RNAP at the +1 site (start site) Promoters: 2 important sequences part of promoter region - 35 and -10 region (Pribnow box)

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Initiation of RNA chains:


binding of RNAP holoenzyme to promoter region

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1) RNAP holoenzyme binds loosely to -35 region (dsDNA), then tightly to the -10 region of dsDNA (closed promoter) Localized unwinding of the two strands of DNA provides template strand for RNAP and exposes initiation site (+1)
phosphodiester bond forms between first 2 NTPs of RNA chain

3) unwinds dsDNA ( 17 bp) around -10 region

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6) initiates 8-9 bp then factor is released Core loses affinity for promoter

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5) RNAP chooses correct strand to read (template template)

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When cores loses sigma factor it moves away from promoter Transcription bubble 17 bp Core completes elongation RNAP unwinds dsDNA mRNA displaced from back RNA/DNA hybrid exists

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The steps in transcription: Termination

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rho-independent termination rho-

rho

Terminators complementary region (G:C-rich) which forms hairpin loop in the ssRNA RNAP pauses on UUU and falls off. Termination C C C C G G G G

G:C rich area complementarity causes hairpin RNAP pauses on UUU region 3 RNAP Falls off

mRNA 5

UUUU

Terminators exist either: Intrinsic to RNA strand - intrastrand base pairing (rho-independent) Extrinsic - requires an accessory protein - rho to stop (rho-dependent)

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Electron micrograph of transcription

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Promoter
-120 GC -100 GC

Eukaryotes :
-80 CAAT Box -30 TATA Box Initiation of transcription +1 site

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dsDNA

TATA: positions transcription startpoint CAAT: regulates rate of transcription -initiation and how often GC: helps RNAP bind near startpoint

mRNA made in the nucleus usually differing lengths - called heterogeneous RNA Processing begins immediately in nucleus: mRNA is capped mRNA is polyadenylated mRNA has introns removed Process called post-transcriptional modification post-

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RNA processing after transcription produces a mature messenger RNA (mRNA)

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5 Capping Addition of methylated cap at the 5 end. Added by a special capping enzyme, after enzyme, polymerization of the transcripts first few nucleotides (20-30 NT) (20 Methyl transferase then adds methyl group to guanine (7) Critical for efficient translation of mRNA.

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The Ends of Eukaryotic mRNAs: 5- Capping 5-

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Features: Triphosphate bridge 5 to 5 linkage of guanine (reverse orientation) Guanine is methylated 7 position First 2 NTs of RNA can be methylated NO cap coded for in DNA Essential for ribosome to bind to 5-end 5-

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Polyadenylation Addition of poly-A tail to 3 end ( 100-200 As) poly100 NO DNA template for poly-A tail (no strings of Ts) poly RNAP runs past end of gene (no termination sites) Poly-A polymerase (PAP) adds As onto this Polynew 3 end Thought to stabilize mRNA from degradation Aids in efficiency of translation

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Eukaryotes :

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Embedded in transcript is a poly A site

Cuts 11-30 NT downstream of 11poly A site

Uses ATP

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Transcription in Eukaryotes
3 RNA polymerases responsible for copying DNA template
Pol I: Transcribes large ribosomal RNAs (nucleolus) Pol II: Transcribes mRNA Pol III: Transcribes most small RNAs, tRNAs and RNAs involved in processing primary RNA transcripts

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Transcription in Eukaryotes: mRNA modificaton and processing primary transcript is modified in several ways before release into cytoplasm for translation 5 capping polyadenylation removal of intervening sequences (introns)

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Synthesis of primary transcript Addition of 5methyl cap to transcript

Cap used for ribosome assembly during translation iniitiation... 5-5 link..

AAUAAA signal serves as recognition site for cleavage factors and poly A polymerase

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CPSF (cleavage/polyadenylation specificity factor)

AAAAAAA..

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Elements of the Promoter...

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TATA or GoldbergHogness box caat box

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General Transcription Factors interact with the promoter to facilitate binding of RNA polymerase IIe.g. TBP: TATA binding protein (TFIID) is capable of interacting with several factors that are required for the assembly of a RNA productive transcription polymerase complex... Specific Transcription factors can influence the formation of a productive transcription complex... STFs may bind at some distance from the promoterenhancer sequences

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Intron: RNA that is part of the primary transcript which is removed from mature mRNA

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Exon: part of the primary transcript that remains in the mature mRNA Primary transcript contains both introns and exonsintrons must be removed...

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SNRPS: small nuclear ribonucleoprotein particles

Spliceosome

Macromolecular complex for RNA processing

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Cut at intron border... Two exons joined...

lariat formation Cut at intron border Intron removed

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Why Introns?
Exon shuffling hypothesis: exon/intron organization may facilitate evolution of new genes... In some cases, exons of a gene code for a functional domain- a peptide region that assumes a useful conformatione.g. binding a molecule
-globin: heme containing region

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introns allow ability to mix/assemble different functional cassettes into new combinations?

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Evolution of multigene families and gene divergence


Duplications in DNA could allow for genes to diverge and be incorporated into new physiological contexts
e.g. alpha and beta globin gene families (pp. 785-87)...

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Mutation to new (related) function...

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Heterozygote advantage... Gene duplication...

recombination

Favored by selection...

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Genes expressed at different times during development, in different tissues Proteins are similar but have slightly different physiological properties Order of gene expression in development correlated with linear array in chromosomes

Pseudogenes: duplications that are not functional in the genometend to accumulate random mutations

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Summary of Eukaryotic transcription/translation .

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Small RNAs

translation

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Chromatin Structure
Histones are highly conserved proteins that are intimately associated with the DNA in chromatin
small in size carry a large number of basic residues complexed into a particle termed a nucleosome

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Characteristics of Histones from Calf Thymus DNA


Histone Type H1 H2A H2B H3 H4 Lysine 29% 11% 16% 10% 11% Arginine 1% 9% 6% 13% 14% amino acids 215 129 125 135 102 Molecular WT 23,000 13,960 13,775 15,340 11,280

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nucleosome core particle: Octamer of: H2A H2B H3 H4 ~ 200 bp DNA wrapped around twice (7 fold compaction)

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Further compaction can be achieved by formation of a solenoid of nucleosomes...

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H1 required for formation of 30 nm fiber...

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nucleosomes: beads on a string in different stages of condensation...

10 nm fiber

Low salt

30 nm solenoid

Physiological ionic strength

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Higher order folding... loops of DNA are believed to associate with a protein scaffold...

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Loops of DNA

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scaffold

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Mapping of scaffold attachment regions (matrix attachment sites) in Drosophila...

interaction sites for topoisomerase II are also observed in these regions, as are sites that may be involved in transcriptional regulation...

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How does the configuration of chromatin affect gene expression?


It would seem to be difficult to transcribe DNA that was complexed in the nucleosome core; RNA polymerase appears to displace histone octamers during transcription It is possible to distinguish active from inactive chromatin by susceptibility to enzyme digestion

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nucleosomefree regions

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