Transcript Genomes 3/e - Illinois Institute of Technology

Chapter 12:
Synthesis and Processing
of RNA
Copyright © Garland Science 2007
Genome expression includes 2 steps
•
Initiation of transcription. Assembly of
upstream protein complex; this step
determines whether a gene should be
expressed or not.
• Synthesis & processing of RNA.
RNA polymerase synthesizes
mRNA & subsequently processes or
modifies into mature mRNA.
12-1-1. Synthesis
bacterial RNA
transcripts.
Relatively simpler, only
1 RNA polymerase
1 DNA strand (coding
strand) is used as the
template
A-U or C-G base
pairing; addition of 1
base w/removal of 2
phosphates
Figure 12.1 Genomes 3 (© Garland Science 2007)
12-1-1. Cont.
During elongation,
RNA polymerase has
four subunits, two α
(35 kDa) & β+β’ (150
kDa); σ dissociates
after transcription
initiation.
Occupies 30-bp area
(2 transcription
bubbles of 12-14 bp &
8 bp RNA-DNA
duplex).
Figure 12.2-3 Genomes 3 (© Garland Science 2007)
β subunit
Non-template DNA
Template DNA
New RNA
ds DNA
β’ subunit
Covalent bonds between amino acids & RNA or
DNA (not too tight & not to loose); X-ray
crystallography & cross-linking showed RNA
synthesis happens between β & β’ subunits;
synthesis is not at constant rate; has random
pauses <6 msec; causes backtracking.
Figure 12.4 Genomes 3 (© Garland Science 2007)
12-1-1.
Transcription
termination
Thermodynamic favors
either continuation or
dissociation (NOT stop
codons)
Two mechanisms:
intrinsic terminators
(50% of genes, inverted
palindrome + poly As);
Rho dependent (helicase
breaks DNA-RNA base
pairs)
Figure 12.5 & 7 Genomes 3 (© Garland Science 2007)
12-1-2. Control of
termination
Anti-termination by
specific proteins; binds
upstream of operon;
transfers to RNA
polymerase; leads to
ignorance of termination
signals in operon.
Figure 12.8 Genomes 3 (© Garland Science 2007)
12-1-2. Control of
termination
Bacterial mRNA is
translated at the same
time as it is synthesized.
Attenuation as a mean
to regulate mRNA
synthesis; primarily with
operons for amino acid
synthesis; results in
premature termination.
Figure 12.10 Genomes 3 (© Garland Science 2007)
12-1-2. Control of
termination
Attenuation in E. coli
tryptophan operon.
Ribosome binds at different
places & determines
formation of large or small
RNA loops (the smaller is
the termination signal).
Biological significance:
When Trp is plenty,
attenuation prevents
transcription of Trp
biosynthesis genes.
Figure 12.11 Genomes 3 (© Garland Science 2007)
12-1-2. Control of
termination
Attenuation in
Bacillus subtilis
tryptophan operon is
assisted by trp RNA
binding attenuation
protein (TRAP);
determines formation
of large or small RNA
loops (termination
signals).
Figure 12.12a Genomes 3 (© Garland Science 2007)
12-1-2. Control of
termination
Transcript cleavage
proteins (GreA & GreB)
can reposition the
magnesium ion &
stimulate RNA polymerase
cleavage activity &cut off
detached RNA end &
prevent stalling of a
backtracked polymerase.
Figure 12.13 Genomes 3 (© Garland Science 2007)
12-1-3. Processing
of bacterial RNAs
mRNAs of protein-coding
genes are ready-to-use
in bacteria, but tRNA &
rRNA are synthesized as
precursors & need
cuttings.
3 types of rRNA (5S,
16S, & 23S) by sizes by
sedimentation analysis;
cut by RNase III, P & F;
trim by M16, M23, & M5.
Figure 12.16 Genomes 3 (© Garland Science 2007)
2
4
1
3
5
tRNA
processing
includes a
series of
cuttings by
different
RNases
Figure 12.17 Genomes 3 (© Garland Science 2007)
12-1-3. Processing of bacterial RNAs
Final step is chemical modifications; 50 different
types; tRNA modification makes tRNA recognize >1
codons; rRNA is modified by either adding a methyl
group to 2’-OH or converting uridine to pseudouridine.
Figure 12.18 Genomes 3 (© Garland Science 2007)
12-1-4. Degradation of
bacterial RNAs
Transcriptome is responsive to
environment & cell
physiological status; it
constantly changes, therefore,
needs degradation, an
important way to regulate
genome expression.
2 steps: endonuclease cuts
off the hairpin; exonuclease
degrades from 3’ to 5’.
RNases E & P as a protein
complex called degradosome
Figure 12.19 Genomes 3 (© Garland Science 2007)
12-2-1. Synthesis of
mRNAs in eukaryotes
Chemistry is the same as in
bacteria; RNA polymerase II
has equivalent α, β, β’
subunits; synthesis is quite
different (e.g. poly A tail, 5’
cap, splicing, etc);
processing is at the same
time with synthesis;
Promoter clearance is
transition between preinitiation complex and
synthesis; promoter escape
RNA polymerase II moves
away from promoter.
Figure 12.20 Genomes 3 (© Garland Science 2007)
12-2-1. (Cont.)
Initiation
5’ capping occurs
immediately after
initiation. After
synthesis of 30 nt, γphosphate of 5’ nt is
removed; GTP is
attached; 7’-N is
methylated (type 0
cap); type 1 & 2 caps
are also common.
Significance:
exportation from
nucleus & translation
Figure 12.21 Genomes 3 (© Garland Science 2007)
12-2-1. (Cont.) Elongation
Bacteria: a few min to synthesize a gene at rate of
100 nt per min; Mammals: a few hours for a gene at
a rate of 2000 nt per min; why? Introns (e.g.
dystrophin pre-mRNA 2400 kb, 20 hrs to synthesis)
RNA poly II may pause transcription, so it needs
elongation factors (proteins associated with
polymerase) which can modify chromatin structure.
Table 12.1 Genomes 3 (© Garland Science 2007)
12-2-1. (Cont.) Termination
3’ poly A tail: addition of up to 250 As by PolyA
polymerase in most mRNAs; signals in pre-mRNA
are recognized by cleavage & polyA specificity factor
(CPSF) & stimulation factor (CstF), polyA binding
protein (PADP) helps to recruit As. Significance:
mRNA stability & translation.
Figure 12.22 Genomes 3 (© Garland Science 2007)
12-2-1. (Cont.) Regulation of synthesis
3 possible control mechanisms: 1. RNA
polymerase elongation factor (TFIIS) can restart a
stalled synthesis; 2. Kinases can change
phosphorylation status of C terminal domain; 3.
alternative polyadenylation (poly A at 3’ ends).
Figure 12.23 Genomes 3 (© Garland Science 2007)
12-2-2. Processing – Remove introns
Most mammalian genes contain 50 or more introns;
their positions are sometimes identical in related
species (implications in genome evolution); must be
excised before it can function as mature mRNA.
Figure 12.25 Genomes 3 (© Garland Science 2007)
12-2-2. Processing – Remove introns
Most common introns in vertebrates contain
conserved 5’-GU-3’ & 5’-AG-3’ motifs; some also
contain a poly-pyrimidine tract as splicing signals.
Figure 12.26 Genomes 3 (© Garland Science 2007)
12-2-2. Processing – Remove introns
Splicing in 2 steps: 5’ hydroxyl attack by an internal
A & trans-esterification & formation of a loop (lariat
structure); 3’ trans-esterification & joins with an
adjacent exon. Excised intron is degraded.
Figure 12.27 Genomes 3 (© Garland Science 2007)
12-2-2.
Processing –
Remove introns
Splicing errors:
exon skipping (long
distance between
exons) results in an
exon lost in mRNA;
cryptic splice site
(sequence similarity
with the splicing
motifs) results in part
of an exon lost in
mRNA.
Figure 12.28 Genomes 3 (© Garland Science 2007)
12-2-2. Processing
– Remove introns
snRNAs & proteins
(small nuclear
ribonucleoproteins,
snRNPs) involve in
splicing but not
completely clear yet:
commitment complex;
pre-spliceosome
complex; spliceosome
brings 5’ & 3’ in close
proximity & double transesterification occur.
Figure 12.30 Genomes 3 (© Garland Science 2007)
12-2-2. Processing – Remove introns
Alternative splicing: e.g. 35% of human genes
(35,000 or so) have alternative splicing & give
80,000-100,000 different proteins.
Figure 12.32b Genomes 3 (© Garland Science 2007)
12-2-2. Processing – Remove introns
Biological significance of alternative splicing:
Examples: the human slo gene has 35 exons, 8 are
optional; 88=40,320 possible splicing ways but 500
in reality; each specifying a membrane protein with
subtle differences; active in inner ear hair cells to
detect different sound frequencies 20Hz-20,000Hz
(auditory range of human). In fruit fly, alternative
splicing determines sex (recommended reading).
Figure 12.34 Genomes 3 (© Garland Science 2007)
12-2-2.
Processing –
Remove introns
Trans-splicing:
splicing between
different RNA
molecules; happens in
a few organisms; the
leader segment is
called spliced leader
RNA (SL RNA);
plays a role in gene
expression regulation.
Figure 12.35-36 Genomes 3 (© Garland Science 2007)
12-2-3 & 4.
Synthesis &
Processing of
rRNA & tRNA
are recommended
readings.
(p.363-367)
Figure 12.39 Genomes 3 (© Garland Science 2007)
12-2-5. Chemical
modification of
eukaryotic RNAs
Small nucleolar RNAs
involve in pre-rRNA
modification at 5th NT
of D box.
Many snoRNA are
synthesized from
intron RNA after
splicing.
Figure 12.41 Genomes 3 (© Garland Science 2007)
12-2-5. Chemical
modification of
eukaryotic RNAs
Chemical modifications
can change coding
properties & amino acid
sequence, called RNA
editing.
Above: example of human
apolipo-protein (lipid
transportation); deamination
of a cytosine results in a stop
codon & truncated protein.
Figure 12.42 Genomes 3 (© Garland Science 2007)
Biological significance:
rare but important;
generation of antibody
diversity; RNA synthesis;
etiology of viral disease.
12-2-5. Chemical
modification of
eukaryotic RNAs
Other RNA editing:
Pan-editing (extensive
insertion of nucleotides
into abbreviated RNAs to
produce functional
RNAs); Insertional
editing in some viral
RNAs; Polyadenylation
editing in mitochondrial
mRNAs to create
termination codon.
Figure 12.43 Genomes 3 (© Garland Science 2007)
12-2-6.
Degradation of
eukaryotic RNAs
Half life: 10-20 min in
yeast & several hrs in
mammals. How to
control?
Deadenylationdependent decapping
triggered by removal of
poly(A) tail; 5’-3’
exonuclease digestion.
Figure 12.44 Genomes 3 (© Garland Science 2007)
12-2-6.
Degradation of
eukaryotic RNAs
Nonsensemediated RNA
decay (NMD) or
mRNA surveillance
degrades mRNA with
an incorrect
termination codon
(either by mutation
or incorrect splicing);
again, 5’-3’
degradation.
Figure 12.45 Genomes 3 (© Garland Science 2007)
12-2-6.
Degradation of
eukaryotic RNAs
RNA interference
triggered by Dicer
ribonuclease & generate
short interfering RNAs
(siRNAs) of 21-28 bp;
degraded by RNA
induced silencing
complex (RISC); protect
host from RNA virus.
Figure 12.46 Genomes 3 (© Garland Science 2007)
12-2-6.
Degradation of
eukaryotic RNAs
microRNA (miRNA)
interference is initially
synthesis as precursors
(foldback RNAs); then
cut by Drosha into
hairpins; transported
into cytoplasm & cut by
Dicer; silence mRNAs.
Figure 12.48-49 Genomes 3 (© Garland Science 2007)
Anneals to 3’
untranslated region of
target mRNA & interferes
with protein translation.
12-2-6. Transportation
of eukaryotic RNAs
80% of total RNA in nucleus
need to be transported to
cytoplasm for protein
synthesis through nuclear
pore complexes.
The transportation requires
energy (GTP to GDP); 20
different exportins &
importins in human; possibly
triggered by splicing pathway.
Figure 12.50 Genomes 3 (© Garland Science 2007)
Chapter 12 Summary
Bacterial RNA polymerases synthesize RNAs at a
discontinuous rate interspersed by brief pauses
due to structural rearrangements; termination can
be by 2 mechanisms; functional RNAs are
synthesized as precursors & trimed & chemical
modified; degradation is controlled by enzymes.
Eukaryotic mRNAs are capped by 7methylguanosine at 5’ end & poly(A) at 3’ end;
Pre-mRNAs contain introns & spliced w/snRNAs;
alternative splicing enables more transcripts from
one gene; rRNA is chemically modified
w/snoRNAs; in mRNA is less common; diverse
mechanisms of degradation (e.g. RNA silencing).