(RNA Synthesis)
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Table 11-1, p.265
1
Other Forms of RNA
rRNA and tRNA only appreciated later
• All three forms participate in protein synthesis
• All made by DNA-dependent RNA polymerases
• This process is called transcription
• Not all genes encode proteins! Some encode
rRNAs or tRNAs
• Transcription is tightly regulated. Only 3% of
genes in a typical eukaryotic cell are undergoing
transcription at any given moment
• How many proteins is that???
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How Are Genes Transcribed in
Prokaryotes?
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Stages of Transcription
• binding of RNA polymerase holoenzyme at
promoter sites
• initiation of polymerization
• chain elongation
• chain termination
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4
Properties of Promoters
• Promoters typically consist of 40 bp region on
the 5'-side of the transcription start site
• Two consensus sequence elements:
• The "-35 region", with consensus TTGACA -
sigma subunit appears to bind here
• The Pribnow box near -10, with consensus
TATAAT - this region is ideal for unwinding -
why?
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How Are Genes Transcribed in
Eukaryotes?
• RNA polymerases I, II and III transcribe rRNA,
mRNA and tRNA genes, respectively
• Pol III transcribes a few other RNAs as well
• All 3 are big, multimeric proteins (500-700 kD)
• All have 2 large subunits with sequences
similar to β and β' in E.coli RNA polymerase,
so catalytic site may be conserved
• Pol II is most sensitive to α-amanitin, an
octapeptide from Amanita phalloides
("destroying angel mushroom")
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Transcription Regulation
in Eukaryotes
6
The TATA box in selected eukaryotic genes. The consensus sequence of a number of such
promoters is presented in the lower part of the figure, the numbers giving the percent
occurrence of various bases at the positions indicated.
Transcription Factors
7
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Table 11-3, p.281
Translation
(Protein Synthesis)
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8
Translation in Motion
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The general structure of tRNA
molecules. Circles represent
nucleotides in the tRNA
sequence. The numbers
given indicate the
standardized numbering
system for tRNAs (which differ
in total number of nucleotides).
Dots indicate places where the
number of nucleotides may
vary in
(a) An overlapping
versus a
nonoverlapping code.
(b) A continuous
versus a punctuated
code.
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Codon-anticodon pairing. Complementary trinucleotide sequence elements align in
antiparallel fashion.
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13
Ribosome Assembly/Structure
• If individual proteins and rRNAs are mixed,
functional ribosomes will assemble
• Atomic structures of large and small subunits
are known
• A tunnel runs through the large subunit
• Growing peptide chain is threaded through
the tunnel during protein synthesis
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Structure of the E. coli ribosomal subunits and 70S ribosome, as deduced by X-ray
crystallography. Prominent structural features are labeled. A and B present views of the 30S
(a) and 50S (b) subunits. These views show the sides of these two that form the interface
between them when they come together to form a 70S subunit (c). (d) is a side view of the 70S
ribosome; the white area represents the region where mRNA and tRNAs are bound and peptide
14
Eukaryotic Ribosomes
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15
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Fig. 30-13a1, p. 1001
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Fig. 30-13a2, p. 1001
16
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Fig. 30-13b1, p. 1002
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Fig. 30-13b2, p. 1002
17
The three tRNA-binding sites on ribosomes. The view shows the ribosomal surfaces that form
the interface between the 30S (a) and 40S (b) subunits in a 70S ribosome (as if a 70S
ribosome has been “opened” Like a book to expose facing “pages”). The A (green), P (blue),
and E (yellow) sites are occupied by tRNAs. The decoding center on the 30S subunit (a) lies
behind the top of the tRNAs in the A and P sites (which is where the anticodon ends of the
tRNAs are located). The peptidyl transferase center on the 40S subunit (b) lies at the lower
tips (acceptor ends) of the A- and P-site tRNAs. (Adapted from Figure 5 in Cate,J.H., et al., 1999. X-
ray crystal structures of 70S ribosome functional complexes. Science 285:2095-2104.)
Prokaryotic Initiation
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18
The structure of E. coli N-
formyl-methionyl-tRNAifMet.
The features distinguishing
it from noninitiator tRNAs
are highlighted.
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More Initiation
• Correct registration of mRNA on ribosome
requires alignment of a pyrimidine-rich
sequence on 3'-end of 16S RNA with a
purine-rich part of 5'-end of mRNA
• The purine-rich segment - the ribosome-
binding site - is known as the Shine-
Dalgarno sequence
• Initiation factor proteins, GTP, N-formyl-
Met- tRNAifMet, mRNA and 30S ribosome
form the 30S initiation complex
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Events of Initiation
• 30S subunit with IF-1 and IF-3 binds mRNA,
IF-2, GTP and f-Met-tRNAifMet
• IF-2 delivers the initiator tRNA in a GTP-
dependent process
• Loss of the initiation factors leads to binding
of 50S subunit
• Note that the "acceptor site" is now poised to
accept an incoming aminoacyl-tRNA
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Fig. 30-18a, p. 1006
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Fig. 30-18b, p. 1006
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Fig. 30-18c, p. 1006
22
The Elongation Cycle
• Protein synthesis is vital to cell function, so
elongation factors are present in significant
quantities (EF-Tu is 5% of total protein in E. coli)
• EF-Tu binds aminoacyl-tRNA and GTP
• Aminoacyl-tRNA binds to A site of ribosome as a
complex with 2EF-Tu and 2GTP
• GTP is then hydrolyzed and the EF-Tu:GDP
complex dissociate
• EF-Ts is a guanine nucleotide exchange factor
(GEF) and it recycles EF-Tu by exchanging GTP
for GDP
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Peptidyl Transferase
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Peptide bond formation in protein synthesis. Nucleophilic attack by the α-amino group
of the A-site aminoacyl-tRNA on the carbonyl-C of the P-site peptidyl-tRNA is facilitated
when 23S rRNA purine A2451 abstracts a proton.
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25
Peptide Chain Termination
• Proteins known as "release factors"
recognize the stop codon at the A site
• Presence of release factors with a nonsense
codon at A site transforms the peptidyl
transferase into a hydrolase, which cleaves
the peptidyl chain from the tRNA carrier
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26
The events in peptide
chain termination.
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27
Electron micrograph
of polysomes:
multiple ribosomes
translating the same
mRNA. (From Franke,
C., et al., 1982. Electron
microscopic
visualization of a
discrete class of giant
translation units in
salivary gland cells of
Chironomus tentans.
The EMBO Journal
1:59-62. Photo courtesy
of Oscar L. Miller,
University of Virginia.)
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The characteristic structure of eukaryotic mRNAs. Untranslated regions ranging between
40 and 150 bases in length occur at both the 5'- and 3'-ends of the mature mRNA. An
initiation codon at the 5'-end, invariably AUG, signals the translation start site.
Eukaryotic Initiation
• Begins with formation of ternary complex of
eIF-2, GTP and Met-tRNAiMet
• This binds to 40S ribosomal subunit:eIF-1A &
elF-3: complex to form the 40S preinitiation
complex
• Note no mRNA yet, so no codon association
with Met-tRNAiMet
• mRNA then adds with several other factors,
forming the initiation complex
• Note that GTP is required!
• Proteins of the initiation complex scan to find
the first AUG (start) codon
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The three stages in
the initiation of
translation in
eukaryotic cells.
(Adapted from Pain, V.
M., 1996. Initiation of
protein synthesis in
eukaryotic cells.
European Journal of
Biochemistry 236:747-
771; and Figure 1 in
Gingras,A-C., et al.,
1999. EIF4 initiation
factors: Effectors of
mRNA recruitment to
ribosomes and
regulators of translation.
Annual Review of
Biochemistry 68:913-
963.))
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The structures of various
antibiotics that act as
protein synthesis inhibitors.
Puromycin mimics the
structure of aminoacyl-
tRNA in that it resembles
the 3′-terminus of a Tyr-
tRNA.
Diphtheria Toxin
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31
Diphtheria toxin catalyzes the NAD+-
dependent ADP-ribosylation of selected
proteins. ADP-ribosylation of the
diphthamide moiety of eukaryotic EF-2.
(Diphthamide=2-[3-carboxamido-3-
(trimethylammonio)propyl]histidine.)
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