Chapter 5

The Path to Gene Expression

An overview of the flow of information in eukaryotic cells

1
Transcription: The Basic Process

I. Transcription is done by DNA-dependent RNA polymerases

(RNA polymerases) in both prokaryotes & eukaryotes
A. They incorporate nucleotides to make RNA from DNA
template one at a time
B. RNA sequence is complementary to one of DNA strands

II. RNA synthesis begins with association of RNA polymerase

with DNA template
A. RNA polymerases bind to sites on DNA called promoters, but
they cannot recognize promoters; require the help of
proteins called transcription factors (TFs)
1. TFs are important in initiation of gene transcription
B. Promoter contains information specifying which strand of
DNA to transcribe & the site at which transcription begins

III. Polymerase moves along DNA in 3' —> 5' direction laying
down complementary RNA in 5' —> 3' direction
A. Polymerase catalyzes
reaction:
RNAn + NTP —> RNAn+1 + PPi
1. Ribonucleoside triphosphate
precursors (NTPs) are
hydrolyzed into nucleoside
monophosphates as polymerized
into a covalent RNA chain
B. Reactions must be irreversible
1. To prevent reverse reaction,
nucleic acid synthesis is coupled
to pyrophosphate hydrolysis that
produces two inorganic
phosphates (Pi), making overall
reaction irreversible; done by
pyrophosphatase

2
C. 1. Bacterial RNA polymerase can
incorporate 50 - 100
nucleotides/sec
2. Most genes are transcribed
simultaneously by numerous
polymerases
Number of RNA polymerase
molecules bound to a phage
DNA template

Transcription in Prokaryotes

I. E. coli has a single type of RNA polymerase - 5 subunits

tightly associated to form core enzyme
A. Purify core enzyme, add to solution of bacterial DNA &
ribonucleoside triphosphates
1. Enzyme binds to DNA & synthesizes RNA, but not the
same RNA as in cell
2. It starts synthesis at inappropriate sites (random sites it
would normally have ignored in vivo)

B. Purify accessory protein called sigma ( ) factor & add it

to mix before polymerase attaches to DNA
1. Transcription starts at right places
2.  attachment increases core enzyme affinity for DNA
promoter sites & decreases affinity for DNA in general
3. Once transcription begins,  leaves & core enzyme
continues synthesis

3
Initiation of transcription
in bacteria

II. Bacterial promoters are located in region of DNA strand

just preceding the RNA synthesis initiation site
A. The nucleotide at which transcription is initiated is called
+1; the preceding nucleotide is –1
B. Those portions of DNA preceding initiation site (toward
template 3' end) are said to be upstream from that site
C. Those portions of DNA succeeding initiation site (toward
template 5' end) are said to be downstream from that
site

III. Similar DNA sequences were seen in association with

genes in roughly same location from gene to gene in
bacteria; sequences are called consensus sequences &
are found just upstream from many bacterial genes
A. DNA sequences just upstream from a large number of
bacterial genes have 2 short stretches of DNA that are
similar from one gene to another (-35 & -10 region)

4
IV. The first stretch is centered ~35 bases upstream from
initiation site; typically has TTGACA sequence
A. The TTGACA sequence is a consensus sequence (the –35
element)
B. The –35 region in promoter is recognized by  factor
associated with the RNA polymerase

V. The second conserved sequence is found at ~10 bases

upstream from the initiation site
A. It has the TATAAT consensus sequence (Pribnow box)
B. Responsible for identifying the precise nucleotide at
which transcription starts

VI. Termination of transcription - specific nucleotide

sequence signals termination

A. Usually the polymerase stops transcription on its own when

it reaches a terminator sequence & releases the completed
RNA chain without additional factors
B. Sometimes, a protein called Rho is needed for termination
of bacterial transcription
1. Rho has enzymatic activity that peels the 3' end of RNA
transcript away from the DNA

5
Transcription and RNA Processing in Eukaryotic Cells

I. Eukaryotic cells have 3 distinct RNA polymerases, each

responsible for synthesizing a different group of RNAs
A. RNA polymerase I - synthesizes the larger rRNAs (28S,
18S, & 5.8S)
B. RNA polymerase II - synthesizes mRNAs & most small
nuclear RNAs (snRNAs & snoRNAs)
C. RNA polymerase III - synthesizes various low MW RNAs
(the various tRNAs, 5S rRNA & U6 snRNA)
D. A major distinction between transcription in prokaryotes
& eukaryotes is the requirement in eukaryotes for a large
variety of accessory proteins or transcription factors
1. TFs play a role in every aspect of transcription process
from binding polymerase to DNA template, to initiation
of transcription, to its elongation & termination
2. They are crucial for the operation of all 3 types of
eukaryotic RNA polymerases

II. All 3 major RNA types (mRNA, tRNA, and rRNA) are
processed before they are mature; they are derived from
precursor RNA molecules that are longer than the final
RNA product
A. Primary (1°) transcript (pre-RNA) is the initial precursor
RNA & it is equivalent in length to the full length of the
DNA transcribed; it is then edited
1. Corresponding segment of DNA from which 1° transcript
is transcribed is called transcription unit
2. 1° transcript is typically short-lived, processed into
smaller, functional RNAs
3. Processing requires variety of small RNAs (90 – 300
nucleotides long) & their associated proteins

6
Ribosomal RNAs (rRNAs) & transfer RNAs (tRNAs)

I. Ribosomal RNAs -> 80% of cell RNA, each ribosome consists

of several rRNA molecules with dozens of ribosomal proteins
A. S value (or Svedberg unit) refers to
sedimentation coefficient of RNA;
the larger S value, the larger size

II. Processing of rRNA precursor –

eukaryotic ribosomes have 4 distinct
rRNAs: 3 rRNAs in the large subunit
(28S, 5.8S, 5S in human); 1 in the
small subunit (18S in human)
A. 28S, 18S & 5.8S rRNAs are carved by
various nucleases from a single 1°
transcript (pre-rRNA)
B. 5S rRNA is synthesized from a
separate RNA precursor

III. Transfer RNAs (tRNAs)

A. Transcribed by RNA polymerase III
1. Ribonuclease P is an endonuclease involved in pre-tRNA
processing in both bacterial & eukaryotic cells
B. All mature tRNAs have the triplet CCA sequence at their
3'end; play key role in protein synthesis (amino acid binding
site)

7
Background Information on mRNAs

I. Machinery used in mRNA transcription

A. RNA polymerase II synthesizes all eukaryotic mRNA
precursors
1. RNA polymerase II is composed of 12 different subunits
& is conserved from yeast to mammals
B. Initiation of transcription by polymerase II occurs in
cooperation with a number of general transcription
factors (GTFs; TFs required for accurate transcription
of a diverse array of genes in different organisms)
C. Promoters for RNA polymerase II lie to 5' side of each
transcription unit
1. A critical portion of the promoter lies between 24 & 32
bases upstream from transcription initiation site

2. This region contains a consensus sequence that is either

identical or very similar to 5'-TATAAA-3' & is known as
the TATA box
3. The TATA box of DNA is the site of assembly of a
preinitiation complex that contains the GTFs & the
polymerase; this complex must assemble before gene
transcription initiates

8
II. Assembly of preinitiation complex – the first step is
binding of TATA-Binding Protein (TBP) that specifically
recognizes the TATA box of eukaryotic promoters

A. TBP is present as a subunit of complex called TFIID

(Transcription Factor for polymerase II, fraction D);
other subunit of TFIID is TAF (TBP Associated Factor)
B. TBP is a universal TF that mediates binding of all 3
eukaryotic RNA polymerases; present as one of the
subunits of 3 different proteins
1. As a subunit of TFIID, TBP promotes binding of RNA
polymerase II
2. As a subunit of the proteins SL1 or TFIIIB, TBP
promotes binding of RNA polymerases I or III

III. TFIID binding sets stage for

assembly of complete
preinitiation complex
A. 3 GTFs interact with promoter on
DNA (TBP of TFIID, TFIIA &
TFIIB) & provide platform for
binding of huge, multisubunit RNA
polymerase with attached TFIIF
1. Once RNA polymerase-TFIIF is
in position, TFIIE & TFIIH joins
the complex & transforms the
polymerase into an active,
transcribing machine
B. TFIIH is the only GTF known to TAF; TBP associated factor
have enzymatic activity TFIIB: provides a binding
1. One of its subunits functions as site for RNA polymerase
TFIIF is bound to RNA
a protein kinase to phosphorylate
polymerase II
RNA polymerase

9
 2. 2 other subunits act as DNA unwinding enzymes
(helicases); the DNA helicases separate DNA strands of
promoter; allows polymerase access to template strand
C. Once transcription starts, certain of the GTFs (including
TFIID) may be left behind at promoter site, while others
are released from the complex
1. As long as TFIID stays bound to the promoter, more
RNA polymerases can attach to the promoter site &
initiate additional rounds of transcription without delay
D. Carboxyl-terminal domain (CTD) of the largest RNA
polymerase II subunit has an unusual structure
1. It consists of 7 amino acid sequence (-Tyr-Ser-Pro-Thr-
Ser-Pro-Ser-) that is repeated over & over (in mammals,
52 repeats)
2. Of the 7 amino acids, serines 2 & 5 are prime candidates
for phosphorylation by protein kinases
3. RNA polymerase II is phosphorylated just after the
initiation of transcription

4. CTD phosphorylation can be catalyzed by kinases,

including TFIIH
5. Polymerase phosphorylation uncouples the enzyme from
GTFs and promoter DNA, allowing enzyme to escape from
preinitiation complex & move down DNA template
6. A polymerase involved in elongation may associate with a
number of large accessory proteins, including ELL, SII &
PTEFb (elongation factors)

10
IV. Structure of mRNAs - mRNAs share certain properties:
A. Contain a continuous nucleotide sequence encoding a
specific polypeptide
B. Found in the cytoplasm
C. Attached to ribosomes when they are translated
D. Most mRNAs have noncoding, untranslated segment
1. Untranslated regions (UTRs) are found at 5' & 3' ends
(5‘-UTR & 3‘-UTR) & contain sequences that have
important regulatory roles
E. Eukaryotic mRNAs have special modifications at their 5' &
3' ends
1. 3' end of nearly all eukaryotic mRNAs has a string of 50
- 250 adenosine residues called poly(A) tail;
2. 5' end – has
methylated guanosine
cap

mRNA Processing
I. 1° transcript is processed in the nucleus to produce mature
mRNA that is transported to the cytoplasm
A. The process requires the addition of 5' cap & 3' poly(A)
tail to the transcript ends & removal of introns (splicing)
II. 5'methylguanosine cap forms after
RNA synthesis begins
A. Once the 5' end of mRNA precursor
is synthesized,
1. Last phosphate of the three
phosphates is removed by
triphosphatase, leaving diphosphate
2. Then, GMP is added in inverted
orientation so guanosine 5' end
faces 5' end of RNA chain, thus the
first 2 nucleosides are joined by
unusual 5'-5' triphosphate bridge
(by guanyltransferase)

11
 3. Guanosine is methylated at position 7 on guanine base
while nucleotide on triphosphate bridge internal side is
methylated at ribose 2' position (methylguanosine cap)
(by RNA methyltransferase)

B. These enzymatic modifications at 5' end of 1° transcript

occur very quickly, while the RNA is still in its very early
stages of synthesis
C. May serve several functions: prevents exonuclease
digestion of mRNA 5' end, aids in transport of mRNA out
of nucleus, important role in initiation of mRNA translation

III. The poly(A) tail – 3' end of mRNA contains a string of

adenosine residues that forms a tail
A. Tail invariably starts ~20 bases downstream from
AAUAAA, a recognition site for a protein complex that
carries out processing reactions at the mRNA 3' end
1. Among proteins of processing
complex, endonuclease cleaves
the pre-mRNA downstream from
the recognition site
2. After nuclease cleavage, poly(A)
polymerase adds ~250 adenosines
without the need of template
3. The poly(A) tail together with an
associated proteins protects the
mRNA from degradation by
exonucleases

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IV. RNA splicing: removal of introns
A. RNA splicing requires break at 5' & 3' intron ends (splice
sites) & covalent joining of adjacent exons (ligation)
1. Splicing process must be absolutely precise because a
single base error (addition or loss) at any of the splice
junctions changes the reading frame & results in mRNA
mistranslation
2. Conserved sequence at eukaryotic exon-intron borders in
mammalian pre-mRNA is G/GU at 5' intron end (5' splice
site) & AG/G at 3' end (3' splice site)

B. These sequences are necessary for splice-site recognition,

but they are not sufficient
1. Additional specific sequences that allow splicing machinery
to distinguish between exons & introns are exonic splicing
enhancers (ESEs) situated within the exons

V. Two types of intron splicing mechanisms have been

described – group I & group II introns

A. Group I introns - common in fungal/plant mitochondria,

plant chloroplasts & in nuclear RNA of lower eukaryotes
B. Group II introns - seen in fungal mitochondria, plant
chloroplasts & bacteria
1. Group II introns undergo self-splicing by passing through
intermediate stage (lariat)
2. First step is cleavage of 5' splice
site followed by formation of
lariat by covalent bond between 5'
end of intron & an adenosine
residue near the 3' end of intron
3. The subsequent cleavage of 3'
splice site releases lariat & allows
cut ends of exons to be covalently
ligated

13
VI. RNA splicing in animal cells

A. RNA splicing in animal cells is similar to Group II introns;

difference is that intron can't splice itself; it needs many
snRNAs (small nuclear RNAs) & their associated proteins

B. Processing occurs as each intron becomes associated with

macromolecular complex (spliceosome)
1. Each spliceosome consists of a variety of proteins & a
number of distinct ribonucleoprotein particles (snRNP, a
complex formed between RNA and RNA-binding proteins)

C. Once spliceosome machinery is assembled, snRNPs cut the

introns out of the transcript & paste the ends of the
exons together

D. Intron removal requires several snRNPs: U1, U2, U5 &

U4/U6 snRNPs

1. U1 snRNP attaches at the 5’

splice site of intron
2.U2 enters the branch point, a
specific adenosine residue,
become the branch point of
lariat
3.U4/U6 and U5 enter and U1
dettaches. U6: ribozyme and
U4 is an inhibitor of catalytic
activity of U6
4.U6 cleaves of 5’ splice site
5.U5 associates with free 5’
exon and 3’ splice site
6.Cleavage of 3' splice site (by
U6) releases lariat & allows
exons to be covalently ligated

14
VII. The exonic splicing enhancers (ESEs) that play a key
role in exon recognition by splicing machinery and serve
as binding sites for RNA-binding proteins called SR
proteins
A. SR proteins help recruit snRNPs to splice sites

Evolutionary Implications of Split Genes and RNA Splicing

I. RNA splicing is one of the steps along path to mRNA

formation that is subject to cellular regulation
A. Many 1° transcripts can be processed by ≥2 pathways so
that a sequence that acts as an intron in one pathway
becomes an exon in an alternate pathway
B. Because of this alternate splicing; the same gene can
code for >1 polypeptide

Small Non-Coding RNAs and RNA Interference

I. RNA interference or double-stranded RNA (dsRNA)-

mediated interference (RNAi)

A. dsRNAs taken up by cells induce a selective destruction of

mRNAs having the same sequence as added dsRNA

B. RNAi was first demonstrated by Fire & Mello (1998); it

can occur in every type of eukaryotic organism
1. It is thought to have evolved as a type of "genetic immune
system" to protect organisms from the presence of
foreign or unwanted genetic material

C. dsRNA blocks formation of a particular protein – the

mechanism

15
1. dsRNA is first cleaved into small
(21–23 nucleotides), double-
stranded fragments (small-
interfering RNAs; siRNAs) by a
ribonuclease called Dicer
2. Following separation, each siRNA
strand becomes associated with
proteins to form a complex that
binds a complementary mRNA
3. Once the siRNA in the complex
has bound to the target mRNA,
the bound mRNA is cleaved by an
associated ribonuclease
4. Each siRNA orchestrates the destruction of many copies
of mRNA, thereby blocking the synthesis of protein
5. We can knock out the gene in question using siRNAs to
destroy the mRNAs & look for cellular deficiencies of the
encoded protein in the laboratory

II. MicroRNAs (miRNAs)

A. Plants & animals produce hundreds of tiny RNAs called

micro RNAs
1. Sizes of miRNAs (at roughly 20 – 25 nucleotides in length)
B. miRNAs are produced by the same processing machinery
responsible for siRNA formation
1. Big difference between siRNA & miRNA - siRNA derived
from transposable element, ds viral product or dsRNA
from researcher, while miRNA is encoded by conventional
genome segment
C. miRNAs turn genes on and off during development
1. Many miRNAs function as translational inhibitors
2. Other potential functions, including gene transcription
suppression, selective mRNA cleavage & induction of
genomic rearrangements

16
Encoding Genetic Information

A. Each amino acid is encoded by 3 sequential nucleotides

(triplet codon)

B. There are 64 (43) possible triplet codons but only 20 amino

acids, what do the extra 44 codons do?
1. Some amino acids are coded for >1 codon
2. 3 codons are termination codons
3. Same amino acids are encoded by different base
sequences

C. Codon assignments are essentially universal (in all living

organisms) regardless of cell type (or organelle)

The genetic code

17
Decoding the Codons: The Role and Structure of the
Transfer RNAs (tRNAs)

I. Protein synthesis is called translation because the language

of nucleic acids must be converted to the language of amino
acids

A. Decoding is accomplished by tRNAs, which act like

adaptors
1. Each tRNA is linked to a specific amino acid (aa-tRNA)
2. Each tRNA is able to recognize a particular codon in
mRNA
3. Interaction between successive codons in mRNA &
specific aa-tRNAs leads to synthesis of polypeptide with
an ordered amino acid sequence

II. tRNAs

1. All tRNAs have relatively small, similar numbers of

nucleotides (73 - 93) & have roughly the same length
2. Have a significant number of unusual bases made by
altering normal base post-transcriptionally
3. Have base sequences in one part of molecule that are
complementary to those in other parts
4. Fold in a similar way to form cloverleaf-like structure with
base-paired stems & unpaired loops
5. Amino acid carried by tRNA is always attached to A
(adenosine) at 3' end of molecule
6. Unusual bases mostly concentrated in the loops where
they disrupt H bond formation in these regions; also serve
as potential recognition sites for various proteins

18
 Psi (ψ): pseudouridine
meG: methlyguanosine
me2G: dimethylguanosine,
I: inosine
D: dihydrouridine

A. The middle tRNA loop has anticodon that H bonds to

complementary mRNA codon, allowing proper amino acid
placement
1. Loop is always made of 7 nucleotides (the middle 3
constitute the anticodon)
2. The same tRNA may be able to recognize > 1 codon
3. Crick's wobble hypothesis – the steric requirement
between the anticodon of tRNA & the codon of mRNA is
very strict for first two positions, but more flexible at
third position
4. 2 codons specifying the same amino acid & differing only
at third position should use the same tRNA in protein
synthesis

19
III. Amino acid activation - each tRNA must attach to
correct (cognate) amino acid
A. Aminoacyl-tRNA synthetases (aaRS) covalently link amino
acids to 3' end of cognate tRNA
1. Each amino acid is recognized by specific aaRS which
charges appropriate tRNAs for amino acid
2. 20 different aaRS, one for each amino acid

IV. Aminoacyl-tRNA synthetases (aaRSs) determine which

amino acid is linked to a particular tRNA
A. Therefore, aaRSs play key role in translation of mRNA
nucleotide sequence into amino acid sequence of
polypeptide

Translating Genetic Information: Background Information

and Prokaryotic Initiation

I. Translation may be the most complex synthetic activity in a

cell
A. It requires a number of cellular constituents:
1. All of the various tRNAs with their attached amino acids
2. Ribosomes
3. mRNA
4. Numerous proteins having different functions
5. Cations
6. GTP
B. Processes are divided into initiation, elongation &
termination

20
II. Ribosome starts translation at a specific mRNA site (the
initiation codon) by attaching there
A. Initiation codon is AUG & by binding there the ribosome
establishes the reading frame
B. Ribosome then moves ahead in consecutive 3 base blocks

III. Translation initiation step 1 in prokaryotes : Bringing the

small ribosomal (30S) subunit to the initiation codon
A. 30S subunit binds to the first AUG (initiation codon)
B. How does 30S subunit select the initial AUG as opposed to
an internal one?
1. Bacterial mRNAs have the specific Shine-Dalgarno (S-D)
sequence, 5 - 10 nucleotides before initiation codon
2. S-D sequence is complementary to sequence near 3' end
of 16S rRNA of 30S subunit
3. Interaction between these complementary sequences on
mRNA & rRNA leads to attachment of 30S subunit at
AUG initiation codon

C. Several steps that require initiation factors (IFs) -

prokaryotic cells require 3 initiation factors, IF1, IF2 &
IF3, which bind 30S subunit & help it attach to mRNA

1. IF1 facilitates attachment of 30S subunit to the mRNA &

prevents the aa-tRNA from entering the wrong site on
the ribosome
2. IF2 is a GTP-binding protein required for attachment of
the first aminoacyl-tRNA
2. IF3 prevents the large (50S) subunit from joining
prematurely to the 30S subunit
& also facilitate entry of
initial aa-tRNA

21
IV. Step 2: Bringing the first aa-tRNA into ribosome - AUG
is initiation codon & the only methionine codon
A. Methionine is always the first amino acid incorporated at
N-terminus of a nascent polypeptide chain
1. In prokaryotes, this initial methionine bears a formyl
group (N-formylmethionine-fMet)
2. Prokaryotic cells have 2 distinct
methionyl-tRNAs: one initiates
(tRNAfMet); the other puts
methionine in interior position
of a polypeptide (tRNAMet)
B. Aminoacylated initiator tRNA
enters the preinitiation complex
at the P site by binding to both
the AUG codon of mRNA & the
IF2 initiation factor; IF1 & IF3
are released

V. Step 3: Assembling the complete initiation complex -

A. Large subunit joins the
complex & the GTP bound to
IF2 is hydrolyzed
B. GTP hydrolysis drives
ribosome conformational
shift required for the
release of IF2-GDP

22
Translating Genetic Information: Eukaryotic Initiation

I. Eukaryotic cells require at least 12 initiation factors (eIFs)

comprising a total of >25 polypeptide chains
A. Several eIFs (eIF1, eIF1A, eIF3) bind to the 40S subunit,
which prepares the subunit for binding to the mRNA

II. The initiator tRNAMet also binds the 40S subunit prior to
its interaction with the mRNA
A. The initiator tRNA enters the P site of the subunit in
association with eIF2-GTP
B. 40S subunit with its associated initiation factors &
charged tRNA form a 43S preinitiation complex and
binds to 5' end of mRNA, bearing the methylguanosine
cap

III. 43S complex is recruited to mRNA with the help of

initiation factors that are already bound to the mRNA
A. eIF4E binds to the 5' cap of the eukaryotic mRNA
B. eIF4A moves along the 5' end of the message removing
any double-stranded regions that would interfere with
the movement of the 43S complex along the mRNA
C. eIF4G serves as linker between 5‘ cap & 3' poly A tail; it
converts a linear mRNA into a circular message
1. The reasons for the circularization of eukaryotic mRNAs
are not entirely clear

23
IV. After the 43S complex binds to mRNA 5' end, the
complex then scans along message until it reaches a
recognizable nucleotide sequence (typically 5'-
CCACCAUG-3'), containing AUG initiation codon
A. Once the 43S complex reaches the AUG codon, eIF2-
GTP is hydrolyzed
B. eIF2-GDP & other associated eIFs are released & the
large 60S subunit joins the complex to complete
initiation

The Role of the Ribosome

I. Ribosomes play major roles in translation

A. Select tRNAs & bind protein factors
B. Catalyze a key reaction, the formation of the peptide
bond (polymerize amino acids)
C. Ensure accurate translation
II. Each ribosome has 3 sites for
association with tRNAs; the sites
receive each tRNA in successive
steps of elongation cycle
A. A (aminoacyl) site - generally
aminoacyl tRNAs enter here
B. P (peptidyl) site - tRNAs donate
amino acid(s) of growing chain from
here
C. E (exit) site - tRNA leaves from here
after losing attached amino acid(s)

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Elongation

I. Step 1 : Aminoacyl-tRNA selection -

after initiation, charged initiator tRNA
is in P site & A site is empty
A. Second aa-tRNA enters vacant A site in
first elongation step
1. Before second aa-tRNA can bind the
mRNA in A site, tRNA must bind with a
elongation factor linked to GTP (EF-Tu
in prokaryotes; eEF1 in eukaryotes)
2. EF-Tu is required to deliver aa-tRNAs
to the A site of the ribosome
3. aa-tRNA-Tu-GTPs with right anticodon can enter A site
4. Once the proper aa-tRNA-Tu-GTP is bound to the mRNA
codon, the GTP is hydrolyzed
5. Then, the Tu-GDP complex is released

II. Step 2: Peptide bond formation

A. Peptide bond forms between these 2 amino acids

1. Amino group of aa-tRNA in A site reacts with carboxyl
group of aa-tRNA in P site; displacing P site tRNA
2. tRNA attached to second
codon in A site now has 2 amino
acids (dipeptide); tRNA in P
site has none (deacylated)
3. Catalyzed by peptidyl
transferase, a component of
the large subunit of the
ribosome

25
III. Step 3: Translocation - requires GTP-bound elongation
factor (EF-G in prokaryotes; eEF2 in eukaryotes) & GTP
hydrolysis
A. Translocation is characterized by dramatic shifts in
position between the ribosome's 2 subunits
1. The ribosome moves three nucleotides (one codon) along
mRNA in 5'—>3' direction
2. Translocation is
accompanied by the
tRNA-dipeptide moving
from A site to P site, the
deacylated tRNA moves
from P site to E site

IV. Step 4: Releasing the deacylated tRNA - uncharged

tRNA leaves P site —> E site —> out of ribosome
emptying the E site
A. Once peptidyl-tRNA moves to P site by translocation,
A site is once again open & new aminoacyl-tRNA moves in
1. Anticodon of new tRNA is complementary to third codon
2. Get new peptide bond (now
3 amino acids [tripeptide]
on tRNA in A site),
translocation & another
open A site, binding of
fourth tRNA, etc.
B. The elongation cycle
continues until termination

26
Termination

I. There are no tRNAs whose anticodons are complementary

to stop codons (UAA, UGA, UAG); protein released when
one is reached
A. Termination requires the release factors that interact
directly with stop codons
B. Bacteria have 3 release factors - RF1 recognizes UAA &
UAG; RF2 recognizes UGA & UAA; RF3 is not codon-
specific, but increases the activity of the other factors
C. Eukaryotes have 2 release factors (eRF1 & eRF3); work
together & recognize all of the stop codons
D. Once translation stops, both the deacylated tRNA & the
release factor are then released
E. Once termination is complete, the ribosome separates
from mRNA & dissociates into its large & small subunits;
they will then be ready for more translation

Polyribosomes

I. Many ribosomes are usually attached to the same mRNA;

can be seen in EM; this complex of mRNA & ribosomes is
called a polyribosome or polysome
A. After first ribosome assembles on chain & moves a
sufficient distance, another attaches & begins
translation
B. Simultaneous translation of mRNA by many ribosomes
greatly increases cell protein synthesis rate

27