Promoters – DNA sequences that provide direction for RNA SEQUENCE OF EVENTS

polymerase

- Promoter region to which RNA pol binds is closer to the

3’ end of the template strand than the actual gene for
the RNA to be synthesized
- RNA formed from 5’3’ end
- Polymerase moves along template strand from 3’5’
end
- Coding strand = 5’3’
- Binding site  upstream (5’ of coding strand; 3’ of the
template strand)

Transcription Start Site (TSS) – first base incorporated into RNA

chain, at position +1

Pribnow Box – first promoter element, -10 region

Core promoter - -35 region/element

UP element – upstream of the core promoter, which enhances

the binding of RNA polymerase; usually extends from -40 to -60

Extended promoter – region from the end of the UP element to

the TSS

Consensus sequences – base sequence of promoter regions that

contain many bases in common
- Promoter regions are A-T rich, with 2 hydrogen
bonds/base pair; more easily unwound than G-C rich
regions, which have 3 hydrogen bonds/base pair
- Promoter base sequence controls the frequency with
which the gene is transcribed
- A strong promoter binds RNA pol tightly  gene is
transcribed more often
CHAIN INITIATION – begins when RNA pol binds to the promoter
& forms a closed complex
- The sigma-subunit directs the polymerase to the
promoter  bridges the -10 and -35 regions of the
promoter to the RNA polymerase core via a flexible “flap”
in the sigma-subunit
- Core enzymes lacking the sigma-subunit bind to areas of
DNA that lack promoters
- Holoenzyme may bind to “promterless” DNA, but it
dissociates without transcribing
- Chain initiation requires formation of the open complex
- Portion of the beta-prime and the sigma-subunits 
initiate strand separation, melting about 14 bp surround
TSS
- Purine ribonucleoside triphosphate  first base in RNA,
binds to its complementary DNA base at position +1
- Adenine tends to occur more often than G
- First residue retains its 5’-triphosphate group
CHAIN ELONGATION
- After the strands have separated, a transcription bubble
of about 17 base pairs moves down the DNA sequence to
be transcribed
- RNA polymerase catalyzes formation of phosphodiester
bonds between the incorporated ribonucleotides
- When about 10 nucleotides have been incorporated, the
sigma subunit dissociates, later recycled to bind to
another RNA polymerase core enzyme
Transcription process supercoils DNA
Negative supercoiling – upstream
Positive supercoiling – downstream
Topoisomerases – relax the supercoils in front and behind the
advancing transcription bubble
- Rate of chain elongation is not constant  RNA pol
moves quickly though some DNA regions
- Abortive transcription – instead of finishing every RNA
chain, RNA pol releases most chains near the beginning
of the process (5-10 nucleotides)  this is due to failure
of RNA pol to break its own bonds to the promoter via
the sigma subunit
- For chain elongation to occur  RNA pol must be able to
launch itself off the promoter
 - Tight binding between the sigma subunit and promoter
 requires substantial energy
- RNA polymerase bound tightly to the DNA promoter 
it “scrunches” the DNA into itself  causing torsional
strain of the separated DNA strands
CHAIN TERMINATION – involves specific sequences downstream
of the actual gene for the RNA to be transcribed
TWO TYPES OF MECHANISMS
1. Intrinsic termination – controlled by termination sites
- Termination sites characterized by 2 inverted repeats
 sequences of bases complementary s/t they can
loop back on themselves
- DNA encodes a series of uracils
- When RNA is created, inverted repeats form a hairpin
loop  tends to stall the advancement of RNA pol
- Presence of Uracils causes series of A-U base pairs bet.
Template strand and RNA
- A-U pairs weakly than G-C  RNA dissociates from the
transcription bubble  ending transcription
2. Rho-dependent - also cause hairpin loop
- Rho protein binds to RNA & chases the polymerase
- When pol transcribes RNA that forms hairpin loop, it
stalls  giving the rho protein a chance to catch up
- When rho protein reaches termination site 
facilitates dissociation of pol-DNA complex
- Movement of Rho + dissociation  requires ATP
TRANSCRIPTION REGULATION IN PROKARYOTES
1. SIGMA FACTORS – viruses & bacteria produce different
sigma-subunits that direct the RNA pol to different genes
a. Phage SPO1 – virus that infects Bacillus subtilis
Early genes – transcribed by the host’s RNA pol; using its
regular sigma-subunit  codes for gp28  another sigma-
subunit  directs RNA pol to transcribe preferentially more
of the viral genes during middle phase
Middle genes – gp33 and gp34  make up another sigma
factor  directs transcription of late genes
- Note: sigma factors are recycled  initially B. subtilis
uses standard sigma factor  as more & more gp28 is
produced, it competes for binding with standard sigma
for RNA pol  eventually subverting transcription for
virus instead of the bacterium
b. Heat shock – normal sigma-subunit is called sigma^70
- When E. coli is grown at high temp than optimum 
produce another set of proteins
- Sigma^32  directs RNA pol to bind to diff. promoters
not normally recognized by sigma^70
2. ENHANCERS – upstream activation sequences (UAS)
- Sequences bound by transcription factors; sequences
that assist in initiation
- Location not fixed, still functions as enhancers
- Usually upstream of the promoter
- Polymerase does not bind to enhancers
- Acts even if located downstream from start of
transcription
- Bidirectional  sequence may be reversed
- Promiscuous  enhance expression of any gene in its
vicinity
UPSTREAM SITES OF GENES FOR rRNA PRODUCTION:
1. Fis sites – binding sites for Fis protein
o Extend from the end of the UP element at -60 to -150
(examples of a class of DNA sequences called
enhancers)
o Enhance transcription of rRNA
3. OPERONS – assemblage of operators, promoter, structural
genes  encode enzymes for metabolic pathways
- Genes not transcribed all the time
- Inducer – triggers production  induction
- Operator – site of regulation
- Promoter – site of interaction w/ RNA pol
Regulatory protein: inducer – turns on operon (induction)
Repressor – turn off (repression)
LAC OPERON – controlled by negative regulation
Lactose – disaccharide, substrate of beta-galactosidase
- Will use lactor/galactose as substrate only in absence of
glucose
- Beta-galactosidase – hydrolyzes the glycosidic linkage between
galactose and glucose (monosaccharide components)
- Glucose  preferred energy and carbon source
- Enzymes that act on galactose are not turned on until cell is
glucose-starved
- Structural genes are not expressed
- Repressor protein is usually expressed
- Protein binds to operator site blocking RNA pol II from binding
to promoter
- Operator site has palindromic DNA sequence
- LacZ  codes for beta-galactosidase
- LacY  lactose permease  allows lactose to enter the cell
- lacA  transacetylase  inactivate certain antibiotics that may
enter the cell via lactose permease
- lacI  regulatory gene  controls gene expression 
responsible for the production of a protein (repressor)
- Repressor – inhibits the expression of structural genes
- Presence of inducer  inhibition is removed
- Inducer – binds to repressor, convert it into a conformation - Genes that lack TATA box have Inr (initiator element)
that’s unable to bind to operator site  structural genes 2. Regulatory elements
transcribed - Bind to transcription factors
CATABOLITE ACTIVATOR PROTEIN (CAP) - Response to physiological signals (hormones)
- cAMP receptor protein - Heat shock
- overrides influence of any inducers
- activate adenylyl cyclase PROMOTERS – complex
- high levels of cAMP
- cAMP induce CAP synthesis TYPES:
CONTROL OF OPERONS 1. TATA box
1. positive control – genes expressed only if regulatory 2. CAAT box
protein is present 3. GC box
2. negative control - Found in “housekeeping genes”
CLASSIFICATION OF OPERONS
1. inducible ENHANCERS
2. repressible 1. Response element – promoter modules that are
responsive to common regulation
4. TRANSCRIPTION ATTENUATION – control mechanism by a. Heat shock element
which transcription is altered before it has begun b. Glucocorticoidal response
c. Metal response element
TRANSCRIPTION IN EUKARYOTES
- More complicated due to presence of histones  nucleosomes 2. Transcription factors for different response elements &
 chromosomes  expression is repressed initiate transcription of proteins
- HSTF bind to HSE
Transcription co-regulators – enzymes that loosen DNA:histone INITIATION OF TRANSCRIPTION
interactions - Formation of eukaryotic transcription initiation complex
- ATP-dependent chromatin remodeling - Requires: Pol II, 5 GTFs, mediator protein (Srb Med)
- Complexed  change shape of chromosome - Process:
THREE CLASSES 1. A TBP (TATA binding protein) bind to TATA
1. RNA Pol I – synthesis major rRA 2. TAF & TBP form complex that serves as bridge
2. RNA Pol II – synthesis mRNA 3. DNA is bent s/t DNA sequence is upstream &
3. RNA Pol III – synthesis tRNA, 5S rRNA, small RNAs downstream to TATA
including those involved in mRNA processing 4. GTFs convene
RNA Pol II 5. Transcription begins
Yeast RNA Pol I  homologous to human RNA Pol II - Eukaryotic transcription depends on:
RNA Pol II – 12 different polypeptides; numbered RPB1 to RPB12, 1. Relief of repression d/t chromatin structure
RPB3 2. Interaction of RNA Pol II with promoter
RPB4 & RPB7  unique to RPII 1. HATs (Histone Acetyl Transferases)
All others shared by RPI, RPII, RPIII - Acetylation of epsilon-NH3+ groups in lysine residues in
histone tails
RPB1 – C-terminal domain (CTD) - Removes (+) charge  destabilizes histone-DNA
27 repeats of YSPTSPS interactions
- Hydrophilic - Works w/ phosphorylation of serine residues
- Contains many sites for phosphorylation (OH grp) - Introduces (-) charge  destabilize histone-DNA
- Essential to RNA Pol II action interaction
HISTONE DEACETYLASE COMPLEXES
TWO SEPARATE SEQUENCES (Eukaryotic Gene Regulation) - Restores (+) charge in histones  restores histone-DNA
1. Core element interaction
- Contains TATA box
- Consensus sequence: TATAAAA
- Usually -25 bp (upstream)
CHROMATIN REMODELLING COMPLEXES
- ATP-dependent conformational
- Essential features – alter nucleosomes
- Interaction of RNA Pol II
ROLE OF MEDIATOR PROTEINS
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DNA recognition by gene regulatory proteins
Principle: structural & chemical complementarities
- H-bonding, DNA sugar-phosphate backbone
- Protein – contact with major groove
3 MOTIFS IN DNA-BINDING PROTEINS
1. HTH (Helix-turn-helix) – dimensions of alpha-helix
- Segment of alpha-helix that fits into major groove
- (2) alpha-helices separated by beta-turn
- Additional interaxn: methylation of nucleobases
- Width of major groove & alpha-helix is the same
- Most common motif
- Sequence of 20 AA that’s relatively conserved in many
different DNA-binding proteins
- 1st helical region  first 8 residues of the region
- 2nd helical region  seq. of 3 or 4 AA
- Position 9  glycine involved in beta-turn
2. Zn-finger - motif gets its name from the shape adopted
by the 12 AA that are looped out from the intersection of
the 2 cysteines and 2 histidines with Zinc ion
- When TFIIIA binds to DNA, the repeated zinc fingers
follow the major groove around the DNA
TWO CLASSES
a. C2H2 – 2 cysteins; 2 histidines
b. Cx
3. Leucine zipper base region (Basic-region Leucine zipper
motif)
- Third major class of sequence-dependent DNA-
binding protein
- Periodic repetition of Leucine residues w/in helical
regions (every 7 residues)
- Dimerize to form y-shaped molecules
- Stem – zipper of leucine residues
- Arm – contain basic regions A & B
- Half of the protein is composed of the basic region,
with many conserved residues of lysine, arginine &
histidine
- Second half contains series of leucines every 7
residues
- Takes 3.6 AA to make a turn of an alpha-helix
- With a seven-residue spacing, the leucines all line up
on one side of an alpha-helix
- Zipper  d/t line of hydrophobic residues interacts
with a second analogous protein fragment via
hydrophobic bonds, interweaving like a zipper
- DNA-binding proteins w/ leucine zippers bind the
DNA in the major groove via the strong electrostatic
interactions between the basic region & sugar
phosphates
- Protein dimers form & the leucine half interacts with
the other subunit, while the basic part interacts with
the DNA
TRANSCRIPTION-ACTIVATION DOMAINS
Motifs whereby transcription factors recognize other proteins
1. Acidic domains – regions rich in acidic AA
Gal4  transcription factor in yeast that activates genes
for metabolizing galactose
- Domain of 49 AAs, 11 of which are acidic
2. Glutamine-rich domains - seen in transcription factors
Sp1  upstream transcription factor that activates
transcription in the presence of an additional promoter
element (GC box)
- It has 2 glutamine-rich domains, one of which contains
39 glutamines in 143 AAs
- CREB & CREM have this domain
3. Proline-rich domain – seen in activator CTF-1
- Domain of 84 AA, 19 of which are prolines
- CTF-1  member of a class of transcription factors that
bind to an extended promoter element (CCAAT box)
- N-terminal domain regulate transcription of genes
- C-terminal end  transcription regulator; bind to
histone proteins via proline repeats
INDIRECT READOUT
- DNA sequence produces a unique contour of dsDNA that
proteins can recognize
- Proteins usually interact
- From the major groove
- With part of nucleobases not involved in H-bonding
POST-TRANSCRIPTIONAL RNA MOD
1. tRNA & rRNA
Trimming and addition of terminal sequences – produce
tRNAs with proper size & base sequence
- tRNA contains a CCA sequence at 3’ end  presence of
this portion is important d/t 3’ end is the acceptor for AA
to be added to growin protein chain
- Trimming  nucleus
- Methylation  cytosol
- Processing of rRNA  methylation & trimming to proper
size
- base modification  Methylation & substitution of sulfur
for oxygen
- 3 rRNAs  sedimentation coefficient of 70S
- Smaller subunit  SC of 30S, one RNA mol sc of 16S
 - 50S subunit contains 2 kinds of RNA  sedimentation
coefficient of 5S & 23S
- Eukaryotic ribosomes  sedimentation coefficient of
80S, with 40S & 60S subunits
- 40S subunit contains an 18S RNA & the 60S subunit
contains a 5S RNA, a 5.8S RNA & 28S RNA
2. Messenger RNA
- Extensive processing happens in eukaryotic mRNA
- Mods include:
a. capping of 5’ end
b. polyadenylating (adding a poly-A sequence) to the 3’
end
c. splicing of coding sequences
- processing is not a feature of prokaryotic mRNA synthesis
POST TRANSCRIPTIONAL MOD OF Mrna
- the cap at the 5’ end of eukaryotic mRNA is guanylate
residue  methylated at N-7 position
- methylated guanylate residue  attached to
neighboring residue by 5’-5’ triphosphate linkage
- 2’-hydroxyl group of the ribosyl portion of neighboring
residue  frequently methylated
- Polyadenylate tail (poly-A) at 3’ end of a message (100-
200 nucleotides long)  added before the mRNA leaves
the nucleus
- Poly-A tail  protects the mRNA from nucleases &
phosphatases which degrades it
- Adenylate residues are cleaved off before the portion of
the molecule that contains the message is attacked
- Presence of 5’ cap  protects the mRNA from
exonuclease degradation
- Prokaryotic genes  continuous
- Eukaryotic genes  discontinuous d/t intervening
sequences that do not appear in the final base sequence
of the mRNA for the gene product
SPLIT GENES – introns and exons
1. INTRON –noncoding; intervening sequences that are not
expressed
- Removed, linking the exons together
- 3’ end is modified by adding a poly-A tail & 7-mG cap 
this yields a matura mRNA
- Actin (muscle protein)  1 intron
- Alpha & beta chains of Hb  two introns for both
- Lysozyme  3 introns
2. EXON – DNA sequences that are expressed or the one
retained in final mRNA product
POST TRANSCRIPTIONAL PROCESSING
Removal of intervening sequences takes places in the nucleus 
RNA forms ribonucleoprotein particles (RNPs) through
association with a set of nuclear proteins
Ribonucleoprotein particles (RNPs)
- Interact with RNA as it’s formed, keeping it in a form that can
be accessed by other proteins and enzymes
- Substrate for splicing  capped, poly-adenylated pre-mRNA
- Splicing requires cleavage at 5’ and 3’ end of introns & joining
of the two ends
- Splice sites - sequence with GU at 5’ end, AG at 3’ end
- Branch sites – within the intron; conserved sequence
- Found 18 to 40 bases upstream from the 3’ splice site
- PyNPyPuAPy (Py – pyrimidine; Pu – purine, N – any
nucleotide, A is invariant)
- G  always present on 5’ end of intron lopps back in
close contact with the invariant A from the branch point
- 2’ OH of the A  performs nucleophilic attack on
phosphodiester backbond at 5’ splice site  forming
lariat structure & releasing exon 1
- AG at 3’ end of exon, does the same to G at 3’ splice site
 fusing two exons
Small nuclar ribonucleoproteins (snRNPs)
- Splicing depends on snRNPs to mediate process
- Basic type of RNA  contain both RNA & proteins
- RNA portion  between 100-200 nucleotides + 10 or
more proteins
- One of the most abundant gene products
- Enriched in Uridine residues
- Have internal consensus sequence of AUUUUUG
- Bind to RNAs being spliced via complementary regions
between the snRNP & branch and splice sites
- Certain snRNPs stimulate transcription elongation
- Some RNAs catalyze self-splicing
Spliceosomes – 50S to 60S particle involved in splicing
CAPPING & METHYLATION SPLICEOSOME
ALTERNATIVE RNA SPLICING
1. Constitutive splicing – every
2. Alternative (combinational) splicing – gene expression
- Produce different protein isoforms
Tau protein  accumulates in the brain of Alzheimer’s
patients
- Has 6 isoforms generated by different splicing  forms
appearing during specific devt stages
Troponin T gene  produces muscle protein with isoforms
 RIBOZYMES – catalytic RNA  catalyzes own self-splicing
1. Group I – requirement for external guanosine 
becomes covalently bonded to the splice site in excision
- Transesterification (of phosphoric acid esters) releases
one end of the intron
- Free 3’-OH end of the exon attacks the 5’ end of the other
exon  splicing the two exons & releasing the intron
- Free 3’-OH end of the intron then attacks a nucleotide 15
residues from 5’ end  cyclizing the intron & releasing a
5’ terminal sequence
- Precision of reaction  depend on folded conformation
of RNA  which remains internally H bonded
- Catalytic RNA can act many times  regenerated

2. Group II – display lariat mechanism; no req’t for external

nucleotide; the 2’-OH of internal adenosine attacks the
phosphate at 5’ splice site
- DNA can’t self-splice because it doesn’t have a 2’-OH
- Folding of RNA crucial to catalytic activity, as is with
protein catalysts
- Divalent cation (Mg2+, Mg2+) is required  metal ions
stabilize the folded structure by neutralizing negative
charges on the phosphate groups of the RNA
SELF-SPLICING OF pre-RNA of ciliate protest Tetrahymena
1. A guanine nucleotide attacks at the splice site of the exon
 giving a free 3’-OH end
2. Free 3’-OH end of the exon attacks the 5’ end of the exon
on the right  splicing the two exons & releasing the
intron
3. Free 3’-OH end of the intron then attacks a nucleoside 15
residues from the 5’ end  cyclizing the intron &
releasing a 5’ terminal sequence
 TRANSLATION –
1. Amino acid activation – aminoacyl-tRNA synthetase
AA + tRNA  aminoacyl-tRNA
- AA covalently bonded to tRNA
2. Chain Initiation – first aminoacyl-tRNA is bound to
mRNA at the site that encodes the start of
polypeptide synthesis
Ribosome + mRNA + 1st aminoacyl-tRNA
mRNA and ribosome bound to each other
3. Chain Elongation – peptide bond formed between
amino acids; repeats itself until polypeptide chain is
formed
2nd aminoacyl tRNA brought to ribosome  peptide
bond formed
4. Chain Termination –
THE GENETIC CODE
tRNA – carry AA to site of protein synthesis
mRNA – contain information of AA sequence
tRNA – interacts with mRNA through anticodon loop
a. Triplet – sequence of three bases (codon)
b. Nonoverlapping – no bases shared between consecutive
codons; ribosome moves along the mRNA three bases at
a time
c. Commaless – no intervening bases exist between codons
d. Degenerate – more than one triplet can encode the same
amino acid
e. Unambiguous – only one AA for each codon
f. Universal code -
DETERMINATON OF GENETIC CODE
1. Synthetic mRNA + NTPs  polypeptide
UUUUUU  polyphenylalanin
UUU  Phenylalanine
AAAAA  Polylysine
AAA  Lysine; CCC  Proline; GGG  glycine
2. Filter-binding assay – various tRNA molecules, one of
which is labeled with Carbon-14 are mixed with
ribosomes and synthetic trinucleotides that are bound to
a filter
- Mixture of tRNAs is passed through a filter, and some
bind and others pass through
- If radioactive label is detected on the filter, then it is
known that the particular tRNA did bind
- If radioactive label is found in a solution that flowed
through the filter, then the tRNA did not bind
- This depends on the fact that aminoacyl-tRNAs bind
strongly to ribosomes in the presence of the correct
trinucleotide
- Trinucleotide plays to role of an mRNA codon
- EX: if the aminoacyl-tRNA for histidine binds to the
ribosome in the presence of the trinucleotide CAU, the
sequence CAU is established as a codon for His
CODON-ANTICODON PAIRING AND WOBBLE
- Codon forms bp with complementary anticodon of tRNA
when an AA is incorporated during protein synthesis
- Some tRNAs bond to one codon exclusively, but many of
them can recognize more than one codon d/t variations
(wobble) in the allowed pattern of hydrogen bonding
- Wobble  applies to first base of anticodon
Possibilities of codon-anticodon bp
1. Highly specific? All 61 tRNAs must be present
2. Not strictly followed?
-A poly(U) sequence binds all Phe-tRNAPhe including UUC
(specific to Phe)
- Yeast tRNAAla bind GCU, GCC, GCA
Therefore: Variations in pairing for 3rd bases are allowed
Trp & Met  only one codon
- Single AA can have as many as six codons, as is the case
with leucine & arginine
- Codons sharing the same first letter often code for AA
that are products of one another or precursors of one
another
CODON USAGE and RARE Trna
- Some codons are used more than others (i.e. Leu, Pro,
Ala, Lys, Glu)
- Use of rare tRNAs result to delay in translation
“Wobble” Hypothesis  third base; buffer against deleterious
mutations
- 1st two bases  pair with canonical partners
- 3rd base  “Wobble interaction”
Therefore: Not all tRNAs have to be present
- Any mutation in the third base would not change the
amino acid
- Silent mutation  mutation that doesn’t change
- When the wobble base of the anticodon is uracil  can
base pair not only with adenine, but also with guanine
- When wobble base is guanine  can bp with cytosine &
also with uracil
- Purine base hypoxanthine frequently occurs in the
wobble position in many tRNAs  can bp with ACU in the
codon
- A and C do not form any bp other than the expected ones
with U and G, respectively
- So, when the wobble position is occupied by I (from
inosine, nucleoside made up of ribose & hypoxanthine),
G, or U, variations in H bonding are allowed
CONSEQUENCES OF WOBBLE BASE PAIRING
1. Fewer tRNAs are needed at a given time – other tRNAs in
same family may substitute for missing tRNA
2. Misreading a base may not affect the AA incorporation in
the protein
3. Mutations  may or may not alter the protein
ROLE OF AMINOACYL-tRNA SYNTHETASES – need for activation
of AA and aminoacyl-tRNA
1. AA forms covalent bond to an adenine nucleotide 
producing an aminoacyl-AMP
2. Free energy of ATP hydrolysis  provides energy for
bond formation
3. Aminoacyl moiety is then transferred to tRNA  forming
an aminoacyl-tRNA
Aminoacyl-AMP  mixed anhydride of a carboxylic acid +
phosphoric acid
- Anhydrides  reactive compounds  free energy
change for the hydrolysis of aminoacyl-AMP favors the
2nd step of overall rxn
- Another point that favors the process  energy released
when pyrophosphate (PPi) is hydrolyzed to
orthophosphate (Pi)  to replenish phosphate pool
AMINO ACID ACTIVATION
1. AA + ATP  aminoacyl-AMP + PPi
*anhydride bond bet. Carboxylate + phosphate
2. Aminoacyl-AMP + tRNA  aminoacyl-tRNA* + AMP
*ester bond bet. Carboxylate + 3’OH of ribose
NET: AA + ATP + tRNA  aminoacyl-tRNA + AMP + PPi
TWO CLASSES OF AMINOACYL-tRNA SYNTHETASES
1. Class I – AA is added to 2’OH first before transfers to 3’OH
2. Class II – uses 3’ OH
Synthetase  requires Mg2+ and is highly specific both for the
AA and for tRNA
SECOND GENETIC CODE – two-stage rxn allows for selectivity to
operate at two levels: that of AA and tRNA
SELECTIVITY AT TWO LEVELS
1. Aminoacyl-AMP remains bound to the enzyme
EX: isoleucyl-tRNA synthetase can form an aminoacyl-
AMP of isoleucine or structurally similar Valine
- If the valyl moiety is transferred to tRNA for isoleucine, it
is detected by an editing site in tRNA synthetase 
hydrolyze the incorrectly acylated aminoacyl-tRNA
- Selectivity resides in tRNA, not AA
2. Depends on the selective recognition of tRNAs by
aminoacyl-tRNA synthetases
- Specific binding sites on tRNA are recognized by
aminoacyl-tRNA synthetases
- Exact position of the recognition site varies with different
synthetases
PROKARYOTIC TRANSLATION
Ribosomal Architecture
- Protein synthesis requires the specific binding of mRNA
and aminoacyl-tRNAs to the ribosome
- Ribosomes have specific arki that facilitates the binding
- 30S & 50S – small & large subunits
CHAIN INITIATION
- Synthesis of polypeptide chain starts at N-terminal
- Chain growth  N-terminal to C-terminal
- Coding strand & mRNA read from 5’3’
-