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Transcription

(RNA Synthesis)

Chemistry 40

Chemistry 40
Table 11-1, p.265

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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?

Only a single RNA polymerase


• In E.coli, RNA polymerase is 465 kD
complex, with 2 α, 1 β, 1 β', 1 σ
• β' binds DNA
• β binds NTPs and interacts with σ
• σ recognizes promoter sequences on DNA
• α subunits appear to be essential for
assembly and for activation of enzyme by
regulatory proteins

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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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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?

Chemistry 40

The nucleotide sequences of representative E. coli promoters. (In accordance with


convention, these sequences are those of the non-template strand where RNA polymerase
binds.) Consensus sequences for the -35 region, the Pribonow box, and the initiation site are
shown at the bottom. The numbers represent the percent occurrence of the indicated base.
(Note: The -35 region is only roughly 35 nucleotides from the transcription start site: the
Pribnow box [the -10 region] likewise is located at approximately position -10.) In this figure,
sequences are aligned relative to the Pribnow box.

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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

• More complicated than prokaryotes


• Chromatin limits access of regulatory
proteins to promoters
• Factors must reorganize the chromatin
• In addition to promoters, eukaryotic genes
have ‘enhancers’, also known as upstream
activation sequences
• DNA looping permits multiple proteins to
bind to multiple DNA sequences
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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

• The three polymerases (I, II and III) interact


with their promoters via so-called
transcription factors
• Transcription factors recognize and initiate
transcription at specific promoter sequences
• Some transcription factors (TFIIIA and
TFIIIC for RNA polymerase III) bind to
specific recognition sequences within the
coding region
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Table 11-3, p.281

Translation
(Protein Synthesis)

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Translation in Motion

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What Is the Genetic Code?


• The genetic code is a triplet code
• Codons specify amino acids
• All the codons have meaning
• The genetic code is degenerate
• The genetic code is 'universal'"

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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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How Is an Amino Acid Matched with Its


Proper tRNA?

• A second genetic code exists


• Aminoacyl-tRNA synthetases interpret
the second genetic code
• Aminoacyl-tRNA synthetases
discriminate between the various tRNAs
and amino acids

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What Are the Rules in Codon-Anticodon


Pairing?

• F. Crick's wobble hypothesis for


codon:anticodon pairing
• Some codons are used more than others
• Nonsense suppression occurs when
suppressor tRNAs read nonsense codons

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Codon-anticodon pairing. Complementary trinucleotide sequence elements align in
antiparallel fashion.

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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

Chemistry 40

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

bond formation occurs.


The tunnel through the
50S subunit that the
growing peptide chain
transits is shown as a
dashed line. The
approximate
dimensions of the 30S
subunit are 5.5 X 22 X
22 nm; the 50S subunit
dimensions are 15 X 20
X 20 nm. (Adapted from
Figures 2 and 3 in Cate,
J.H., et al., 1999. X-ray
crystal structures of 70S
ribosomal functional
complexes. Science
285:2095-2104.)

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Eukaryotic Ribosomes

• Mitochondrial and chloroplast


ribosomes are quite similar to
prokaryotic ribosomes, reflecting their
prokaryotic origin
• Cytoplasmic ribosomes are larger and
more complex, but many of the
structural and functional properties are
similar
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What Are the Mechanics of mRNA


Translation?

• All protein synthesis involves three phases:


initiation, elongation, termination
• Initiation involves binding of mRNA and initiator
aminoacyl-tRNA to small subunit, followed by
binding of large subunit
• Elongation: Movement of ribosome along mRNA
and synthesis of all peptide bonds - with tRNAs
bound to acceptor (A) and peptidyl (P) sites.
• Termination occurs when "stop codon" reached

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Fig. 30-13a1, p. 1001

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Fig. 30-13a2, p. 1001

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Fig. 30-13b1, p. 1002

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Fig. 30-13b2, p. 1002

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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

• The initiator tRNA is formylated


methionine: f-Met-tRNAiMet
• A formyl transferase adds the formyl group
N-formyl-Met-tRNAiMet is only used for
initiation, and regular Met-tRNAmMet is
used for incorporation of Met internally
• N-formyl methionine is first aa of all E.coli
proteins, N-terminal Met is removed post-
translationally in many proteins

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The structure of E. coli N-
formyl-methionyl-tRNAifMet.
The features distinguishing
it from noninitiator tRNAs
are highlighted.

Methionyl-tRNAifMet formyl transferase catalyzes the transformylation of


methionyl-tRNAfMet using N10-formyl-THF as formyl donor. The tRNA for reading
Met codons within a protein (tRNAMet) is not a substrate for this transformylase.

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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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Various Shine-Dalgarno sequences recognized by E. coli ribosomes. These sequences lie


about 10 nucleotides upstream from their respective AUG initiation codon and are
complementary to the UCCU core sequence element of E. coli 16S rRNA. G:U as well as
canonical G:C and A:U base pairs are involved here.

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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

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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

• This is the central reaction of protein synthesis


• 23S rRNA is the peptidyl transferase!
• The "reaction center" of 23S rRNA is
composed of bases that are among the most
highly conserved in all of biology.
• Translocation of peptidyl-tRNA from the A site
to the P site follows

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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.

The Role of GTP Hydrolysis

• Two GTPs are hydrolyzed for each amino


acid incorporated into peptide.
• Hydrolysis drives essential conformation
changes
• Total of four high-energy phosphate bonds
are expended per amino acid residue added
- two GTP here and two in amino acid
activation via aminoacyl-tRNA synthesis

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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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The events in peptide


chain termination.

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The events in peptide
chain termination.

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The ribosome life


cycle. Note that IF-3
is released prior to
50S addition.

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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.)

How Are Proteins Synthesized in


Eukaryotic Cells?

• Note the 5'-methyl-GTP cap and the poly A tail


• Initiation of protein synthesis in eukaryotes
involves more than a dozen eukaryotic
initiation factors
• The initiator tRNA that carries only Met and
functions only in initiation - it is called tRNAiMet
but it is not formylated

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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.))

Inhibitors of Protein Synthesis


Two important purposes to biochemists
• These inhibitors have helped unravel the
mechanism of protein synthesis
• Those that affect prokaryotic but not eukaryotic
protein synthesis are effective antibiotics
• Streptomycin - an aminoglycoside antibiotic -
induces mRNA misreading. Resulting mutant
proteins slow the rate of bacterial growth
• Puromycin - binds at the A site of both
prokaryotic and eukaryotic ribosomes, accepting
the peptide chain from the P site, and terminating
protein synthesis
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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

An NAD+-dependent ADP ribosylase


• One target of this enzyme is EF-2
• EF-2 has a diphthamide sidechain
• EF-2 is no longer functional in protein
synthesis following toxin-mediated ADP-
ribosylation, but it still binds GTP
• One toxin molecule ADP-ribosylates many
EF-2s, so just a little is lethal!

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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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