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Transcription
6/8/12
Transcription-synthesis of RNA from DNA template
Performed by RNA polymerase
Holoenzyme made of 5 components in prokaryotes
2’
Three parts to replication: initiation, elongation, termination
Initiation-getting RNA Polymerase to bind to DNA at appropriate locations
Cis-acting sequences on chromosome direct initation- Promoters
Prokaryotes -10 and –35 sequences
TATAAT and TTGACA
Eukaryotes TATA Box and CAAT box
TATA box=AT rich sequence similar to –10;
CAAT box=GGCCATTCT within 100 bases of start site

is responsible for determining initiation site in prokaryotes
alternate  subunits promote binding at variable sites
example: stress response
transcription factors bind to site of initiation and then to RNAP II in eukaryotes
Initiation also involves unwinding of DNA, removal of supercoiling, or chromatin
restructuring before RNA Polymerase can bind.
Elongation-polymerizing complementary ribonucleotide bases into an RNA molecule
Similar in prokaryotes and eukaryotes
In prokaryotes  dissociates after approximately 8 bases have been added
Remaining holoenzyme continues elongation
Move along DNA strand 3’-5’
Synthesize RNA strand from 5’-3’
First base added is at 5’ end
Most recently added base is at 3’ end
Incorporates uracil instead of thymine in RNA molecule
Termination-polymerase stops synthesizing RNA and falls off of DNA strand
Newly formed RNA folds back on itself and makes a hairpin structure
Hairpin may be bound by rho protein complex (rho-dependent termination)
Hairpin structure helps RNA Polymerase dissociate from DNA strand
In Eukaryotes mRNA undergoes maturation and splicing
Takes place in nucleus
5’ cap and 3’ poly A tail are added
splicing occurs
introns removed and exons are spliced together
Translation
In Eukaryotes mRNA undergoes maturation and splicing
Takes place in nucleus
5’ cap and 3’ poly A tail are added
splicing occurs
introns removed and exons are spliced together
Translation-using mRNA template to synthesize protein
Same three steps involved-initiation, elongation, and termination
Genetic code is read so three nucleotides (codon) encode a single amino acid
Genetic code found on page 314 of text book
Has wobble-in many cases the first two bases of a codon determine the
amino acid and the third is not essential (ACX=threonine)
Proteins begin with formyl methionine (fMet) with AUG codon
Three termination codons in code UAG, UGA, UAA
Ribosomes, mRNA and tRNA are key players
Ribosomes made of two subunits
60S and 40S in eukaryotes
50S and 30S in prokaryotes
each comprises small proteins and rRNA
have 3 sites:A-aminoacyl, P-peptidyl, E-exit
tRNA has cloverleaf structure
anticodon on loop will bind to complementary codon on mRNA
amino acid binding site at 3’ end
appropriate amino acid is added by aminoacyl tRNA synthetase
separate enzyme for each amino acid (20 total)
Initiation-Ribosome binds to mRNA near AUG codon (ribosome binding site)
Small ribosomal subunit, fMet tRNA, and mRNA form initiation complex
tRNA binds at P site of ribosome and is positioned at AUG codon
Large subunit then comes to complete the complex
Elongation-addition of new amino acids that are encoded by codons on mRNA
Charged tRNA with anticodon sequence that is complementary to codon moves
into A site
The fMet forms peptide linkage to a.a. at A site
The fMet is released by tRNA and is transferred to A site
A site now has dipeptide
Ribosome moves over one codon
Moves uncharged tRNA to E site from which it leaves
Moves dipeptide bound to tRNA to P site
Opens A site for a new charged tRNA to enter
Process repeats as long as codon at A site has complementary tRNA
Termination-protein synthesis halts and ribosome dissociates from mRNA
Happens when codon for which there is no tRNA enters A site
Termination codons have no corresponding tRNA molecules
May also happen prematurely when amino acid starvation occurs
RNA Polymerase and ribosomes don’t respond to the same signals.
Ribosome doesn’t begin it’s job at transcription start site.