Download - Day 3 - Transcription and RNA Processing
-
7/30/2019 Day 3 - Transcription and RNA Processing
1/50
Transcription and RNA Processing
The Central Dogma
For the phenotypic expression of any gene, information contained inDNA is first transcribed into an RNA from where it is translated intothe amino-acid sequence of the corresponding protein. Phenotypesare the manifestation of the activity/function of proteins and
catalytic RNAs.
DNA RNATranscription
RNA Pol + factors
ProteinTranslation
Ribosomes, tRNAaa+ other factors
Function
Enzyme, structuralelement, hormone,
regulatory, etc
The assumptions of the central dogma aresound, but there are exceptions!
Phenotype
-
7/30/2019 Day 3 - Transcription and RNA Processing
2/50
The Revised Central Dogma
DNA
REVERSE
TRANSCRIPTION Ribozymes
FUNCTION
FUNCTION
Interactions
-
7/30/2019 Day 3 - Transcription and RNA Processing
3/50
Exceptions to the Central Dogma
1. Information contained in RNA can be copied into DNA by reversetranscriptases and telomerase. EXAMPLES: retroviruses, mobileintrons.
2. After transcription, nucleotides can be added to, deleted from, or
modified in some mRNAs by processes known as RNA editing.EXAMPLES: addition and deletion of Us in the mitochondria oftrypanosomes, conversion of Cs to Us in plant mitochondria bydeamination.
3. Not all genes are expressed phenotypically through proteins.EXAMPLES: the RNAs of mobile introns are nucleases, the LSUribosomal RNA has peptide synthetase activity. Such RNAs areclassified as ribozymes.
Ribozymes favor the concept that a more primitiveRNA-based ancestral form of life may have preceded
the DNA-, RNA-, protein-based life as we know it now.
-
7/30/2019 Day 3 - Transcription and RNA Processing
4/50
The first step of the expression of a gene (DNA) is to transcribe theinformation contained in the nucleotide sequence from one strand(template or antisense strand) into an RNA. The product of thistransaction is a transcript.
If a gene carries information for the assembly of a protein, thetranscript is a messenger RNA (mRNA).
The information in mRNAs is decoded by ribosomes and translated byalignment of three-nucleotide codons with corresponding tRNAsthat deliver the corresponding amino acids.
Transcription
Translation
-
7/30/2019 Day 3 - Transcription and RNA Processing
5/50
The messenger: a generalview of transcription and
translation
U UU UU
Coding strand of DNA
Template strand of DNA
mRNA 5 3
-
7/30/2019 Day 3 - Transcription and RNA Processing
6/50
Transcription andRNA ProcessingTopics
1. RNA structure.
2. RNA synthesis.3. Transcription: initiation, elongation and
termination.
4. RNA processing: cleavage of precursortranscripts and modification of bases.
5. RNA and protein splicing.
6. RNA editing.Videos
http://www.hhmi.org/biointeractive/dna/animations.htmlhttp://www.hhmi.org/biointeractive/dna/animations.html -
7/30/2019 Day 3 - Transcription and RNA Processing
7/50
Ribonucleoside triphosphate
O
Base
RNA precursors
Fig. 2.1
RNA polynucleotide chain
5 3
-
7/30/2019 Day 3 - Transcription and RNA Processing
8/50
RNA Structure
Secondary structure: regions of
base pairing (double-strandedhelical regions)
Stem-loop Fig 2.2
Primary structure: order of the nucleotides read 5
35ACUCAUCGGCACGUCAUGCUGAUAUCCGGCUUGACACU 3
Tertiary structure: folding of theentire RNA chain
Pseudoknot
Only non-covalent hydrogenbonding is involved in
secondary and tertiarystructure formation
-
7/30/2019 Day 3 - Transcription and RNA Processing
9/50
Tertiary structure of a transfer RNA
5
3
-
7/30/2019 Day 3 - Transcription and RNA Processing
10/50
Structure ofE. coli16S rRNA
1. ~1500 nucleotides long
2. Compact 3-D folding
3. Highly conserved
5
3
-
7/30/2019 Day 3 - Transcription and RNA Processing
11/50
Kinds of RNA found in a typical bacterium (E. coli)
RNA Function SizeStability
T
No. of
differentkinds
mRNAMessenger contains information forassembly of amino-acid sequence of
protein by ribosomes and tRNA
6-
20S
Few
minutes
~4000
tRNATransfer codon recognition inpartnership with ribosome and transferof amino acid into polypeptide
4S
longerthan one
generation
variable
-
7/30/2019 Day 3 - Transcription and RNA Processing
12/50
RNAs and DNAs can be separated by size in sucrosegradients by centrifugation and by gel-electrophoresis
16S23S30S
30S Precursor
23S16S
5S
+
Start
Separation byElectrophoresis
-
7/30/2019 Day 3 - Transcription and RNA Processing
13/50
Transcription: RNA polymerase (RNAP) catalyzedsynthesis of RNA on a DNA template
Do you remember the principles of nucleic-acid synthesis? Ch. 1!
RNA polymerasesdo NOT require aprimer for theinitiation of RNA
synthesis.
Direction of RNAsynthesis:
5 3
Direction of readingof template DNAstrand:
3
5
-
7/30/2019 Day 3 - Transcription and RNA Processing
14/50
SizeSubunit aa Da Gene Function
alpha () 329 36,511 rpoA Assembly of RNAP, binding of someregulatory proteins, catalysis.
beta () 1342 150,616 rpoB Catalysis of chain initiation and
elongation.
beta () 1407 155,159 rpoC Binding to DNA template.
omega () 91 10,237 rpoZ Restores denatured enzyme to fully
functional form.sigma (70) 613 70,263 rpoD Directs enzyme to corresponding
promoters.
Subunits of the E. coli holo RNAP and
their Functions
-
7/30/2019 Day 3 - Transcription and RNA Processing
15/50
Transcription starts at promoters, located in the DNAupstream of the coding region of one gene or agroup of functionally related genes.
Important Features of Transcription inProkaryotes
Promoters are relatively short nucleotide sequences inthe DNA (genome) that are recognized by sigma ()
proteins (factors). Sigma factors bound topromoters recruit (interact, bind) the RNApolymerase core enzyme for the initiation of
transcription at specific transcription start sites thatare part of the promoter sequence in the DNA.
What do
promoterslook like?
-
7/30/2019 Day 3 - Transcription and RNA Processing
16/50
Typical structure of Bacterial Housekeeping-Gene70
Promoters with 10 and 35 Regions
Question: What do the negative numbers mean?
Fig 2.6
Number of bases upstream (5) of a designated place, which, in thiscase, is the first base of the RNA (transcription start point).
DNA CodingTemplate
RNA (A/G)NNNNN --
53
Coding-strandconsensus sequences
-
7/30/2019 Day 3 - Transcription and RNA Processing
17/50
RNA exitchannel
Core RNA polymerase Holoenzyme
Active site of RNA polymerase and binding of70
Promoter contacts
10 35
pincer
pincerActive sitechannel
Secondary channelfor nucleosidetriphosphate entry
Crabclaw structure
1 4 are domains of70 proteins
-
7/30/2019 Day 3 - Transcription and RNA Processing
18/50
Proteins called factors bind to coreRNAP and direct it to DNA nucleotidesequences called promoters. The twostrands of the DNA are separated by . Atranscription bubble is generated, andtranscription starts at a T or C in thetemplate strand. No primer is requiredfor transcript initiation and no helicase isrequired for the unwinding of the DNA.
RNA synthesis proceeds for ~8 9 ntalong the template, is released.
Transcription starts at promotersand ends at terminators
Fig. 2.7
Transcription continues until thepolymerase with its transcription bubblereaches a transcription termination (t)site. The RNA and the polymerase
dissociate and are released from theDNA.
-
7/30/2019 Day 3 - Transcription and RNA Processing
19/50
Closed complex(RPC)
Open complex(RPO)
binding
Promoter recognition
DNA isomerization
Escape
release and elongation
Termination
AbortInitiation
Transcription elongationcomplex (TEC)
Transcription Cycle
-
7/30/2019 Day 3 - Transcription and RNA Processing
20/50
Not all 70 promoters are created equal
UP element
Extended 10
-
7/30/2019 Day 3 - Transcription and RNA Processing
21/50
Evolutionarily Conserved Regions andFunctional domains of Bacterial 70 Factors
Inhibition of-DNA binding
70 factors initiate the transcription of housekeeping genes (namedafter the 70 kDa housekeeping factor ofE. coli).
Shown below is the linear arrangement of the 70-like factor ofThermus
aquaticus(~43 kDa).
Down arrows indicate contact points with core RNAP proteinsElectrostatic interactionsDirect hydrogen bondsIndirect hydrogen bonds
potential hydrophobic (van der Waals) contacts.
From S. Borukhov and E. Nudler 2003. Curr. Opinion. Microbiol. 6: 93-100
-
7/30/2019 Day 3 - Transcription and RNA Processing
22/50
RNAP Holoenzyme Open Initiation Complex
Template-strand
Coding-strand
Nascent RNA
Part of has been removed to expose the interior of the holoenzymeinitiation complex.
Modified from K.S. Murakami andS. A. Darst 2003. Curr. OpinionStructural Biol. 13: 31-39.
RNA channel
-
7/30/2019 Day 3 - Transcription and RNA Processing
23/50
Transcription Elongation Complex (TEC)
The RNA polymerase adds from 30 to 100 nucleotides per second to thegrowing RNA molecule (6000 maximum per min).
-
7/30/2019 Day 3 - Transcription and RNA Processing
24/50
RNA Polymerase BacktrackingThe formation of a secondary structure (stem-loop or hairpin) at the 3
end of a growing transcript can cause backtracking of the RNApolymerase. The 3 end of the RNA is pushed into the secondary channel.GreA/GreB insert their N-termini, which have exonuclease activity, intothe secondary channel and degrade the RNA until it is rearranged(properly paired with the DNA template?) in the active site channel so that
elongation can be resumed. The true function of RNAP pausing andbacktrackingis somewhatenigmatic.
-
7/30/2019 Day 3 - Transcription and RNA Processing
25/50
The number of different factors
varies greatly among organismsMycoplasma genitaliumhas only ONE factor
E. coli has SEVEN different factorsStreptomyces coelicolorhas MORE THAN SIXTYdifferent factors
Each type offactor recognizes its own uniqueclass of promoter sequences
See nextpage
-
7/30/2019 Day 3 - Transcription and RNA Processing
26/50
The Seven Sigma Factors ofE. coliandtheir Functions
Sigmafactor Gene Function
70 (D) rpoD Principal sigma factor (housekeeping genetranscription).
N (54) rpoN(ntrA, glnF) Nitrogen-regulated gene transcription.
H
(32
) rpoH Heat-shock gene transcription.S (38) rpoS Gene expression in stationary phase cells.
E (24) rpoE Periplasmic stress response proteins.
F (28) rpoF Expression of flagellar operons.FecI fecI Regulates the fecgenes for iron dicitrate
transport.
-
7/30/2019 Day 3 - Transcription and RNA Processing
27/50
70 (D) TTGACA 16 18 TATAAT
H (32) CTTGAA 13 15 CCCCATNT
E (24) GAACTT 15 17 TCTGA
F (28) CTAAA 15 GCCCATAA
FecI (18) GAAAAT 15 TGTCCT
S (38) TTGACA 14 18 CTAYACTT
CONSENSUS SEQUENCESigma 35 region Spacer (b) 10 region
25 region Spacer (b) 12 region
N (54) CTGGCAC 6 TTGCA
Consensus Sequences ofE. coliPromoters
-
7/30/2019 Day 3 - Transcription and RNA Processing
28/50
Transcription is not a smoothly flowing process: pausing occursfrequently at sites that allow the formation ofhairpin structures in theRNA. When hairpins structures form in the portion of the RNA that islocated in the exit channel of the polymerase they cause temporarydisplacement of the 3 OH from the polymerization active site of the RNApolymerase. When this happens, the polymerase backtracks along thetemplate, and several nucleotides at the 3 OH end are pushed into thesecondary channel, where they are progressively removed backward byGreA and GreB until a proper pairing of the RNA with the DNA template is
restored. Pausing is an important feature involved in the regulation ofgene expression through coordination of the synthesis of mRNA with itssimultaneous translation, termination and antitermination oftranscription, and attenuation of transcription.
Watch for signals along the track!
-
7/30/2019 Day 3 - Transcription and RNA Processing
29/50
GC-rich inverted repeat
There are two kinds of transcription termination:1. Factor independent termination and2. Factor-dependent termination.
Transcription Termination
Factor-independent transcription termination occurs at sites in the DNAthat include a region of two-fold symmetry followed by a stretch ofatleast four As in the template strand.
Fig. 2.18MORE
Run ofat least 4As inthe template strand
-
7/30/2019 Day 3 - Transcription and RNA Processing
30/50
RNA
RNA 3 end
Factor-independent transcription termination
Dissociation of nascent RNAand RNA polymerase from
template strand
Fig. 2.18
Folding of the transcript (RNA) in
the active site channel breakshydrogen bonding to thetemplate DNA strand and causesthe release of the RNA and the
core RNA polymerase.
MORE
-
7/30/2019 Day 3 - Transcription and RNA Processing
31/50
Factor-independent transcription termination
How did scientist figure out this mechanism of termination?
Mutations that disrupt base pairing in the hairpin loop structure of RNA(two-fold symmetry in DNA) or shorten the run of adjacent Us (As inthe DNA template) cause continuation of transcription beyondterminator sites.
-
7/30/2019 Day 3 - Transcription and RNA Processing
32/50
Factor-dependent Transcription Termination
Features of factor-dependent transcription-termination sites in DNA: a
site specifying a sequence in the RNA, for example rut, that isrecognized and bound by a protein, for example Rho () that chasesthe RNA polymerase and releases it and the transcript from the DNAtemplate at transcription pausing sites (usually a G:C-rich sequence).
IMPORTANT: Factor-dependent transcription occurs preferentially whenthe translation of a nascent mRNA is stalled.
DNA
Polysomes
DNA
In prokaryotes, translation iscoupled to transcription.
Specifies factor-binding sequence inRNA (EXAMLE:rut)
Any transcription-pause
site in DNA
-
7/30/2019 Day 3 - Transcription and RNA Processing
33/50
Factor-dependent transcription termination
Fig 2.19
How does it work?Rho hexamer binds to rutin thenascent RNA and then chasesthe RNA polymerase until itreaches it at a transcription-pause site on the DNA. Rhothen unwinds the RNA/DNAduplex, releasing the transcriptand the RNA polymerase from
the DNA.
RNA polymerasereaches a pausesite
Stalledribosome
bindsrut
unwindsRNA/DNA hybrid
RNA and RNAP arereleased
-
7/30/2019 Day 3 - Transcription and RNA Processing
34/50
Rho () binds at rutsequences in nascent RNA, moves along RNA(movement requires ATP) until it reaches the RNA polymerase at atranscription-pause site. HOWEVER, if a ribosome has passed a rutsiteBEFORE is bound, then the ribosome prevents from catching up withthe RNA polymerase, and transcription continues past the pause-site.Rho appears to be an RNA/DNA helicase. However, it is not clearwhether or not can directly access the RNA/DNA duplex within theactive-site channel of a paused RNA polymerase core enzyme.
The other two proteins that have termination-factor characteristics similarto those of are:
Tau () and NusA
In comparison to , the RNA-binding sites and interactions ofTau andNusA with the RNA polymerase at transcription pause sites are not wellcharacterized yet.
E. coli has at least three differenttranscription-termination factors
-
7/30/2019 Day 3 - Transcription and RNA Processing
35/50
Antibiotics that Inhibit Transcription
RifampinRifampin is a member of the rifamycins,macrocyclic lactone antibiotics that
inhibit transcription at the initiationstage, but do not block elongation onceinitiation is complete. Rifamycins bindto the -subunit in the wall of theactive-site channel of the RNAP ofbacteria, mitochondria andchloroplasts. Two or three nucleotidesare polymerized.
MORE
-
7/30/2019 Day 3 - Transcription and RNA Processing
36/50
Action of Rifampin
pppApN and pppGpN are the mostcommon products.
The Streptovaricins are related compounds that have the same action as
rifampin, except that they also can block transcript elongation.
-
7/30/2019 Day 3 - Transcription and RNA Processing
37/50
Actinomycin DBinds to the major groove of DNA in G/C rich regions.
Inhibits transcription and replication
Antibiotics that Inhibit Transcription
-
7/30/2019 Day 3 - Transcription and RNA Processing
38/50
Think about it!
How does the process of RNA synthesis differ from DNA synthesis withrespect to:
1. Substrates?
2. Initiation?
3. Template?
4. Priming?5. Ancillary enzymes?
6. Termination?
7. Editing?
-
7/30/2019 Day 3 - Transcription and RNA Processing
39/50
RNA Processing
RNA Modification
and
RNA Editing
i d ( )
-
7/30/2019 Day 3 - Transcription and RNA Processing
40/50
RNA Processing: rRNA and tRNA (rRNA operon)
Transcript (unprocessed precursor RNA)
Fig. 2.20
16 S tRNA 23S 5S
Further processing and modification of bases (maturation)
MATURE rRNAs and tRNAs
Endonucleolytic cleavages by Rnases III, P, etc.
Processed RNA products
SpacerSpacer
-
7/30/2019 Day 3 - Transcription and RNA Processing
41/50
tRNAprocessing
Rnase P
RNase
RNase RNase
rRNAprocessing
in E. coliandB. subtilis
Rnase M5 is similarto type II DNA
topoisomerases
Rnase P consists of an RNA dimer(same sequence, catalytic subunit)
complexed with a dimer of a smallprotein. It is a ribozyme.
difi i
-
7/30/2019 Day 3 - Transcription and RNA Processing
42/50
Structure of a mature tRNA
RNA modification
U
Fig. 2.21
The most common RNAmodification is U
Added by CCAtransferase
IV
II
I
III
Determining base
Dihydrouridine
D d ti f RNA
-
7/30/2019 Day 3 - Transcription and RNA Processing
43/50
Degradation of mRNA
Some of the
enzymesinvolved alsoparticipate inrRNA and tRNAprocessing
Box 2.5
1 1500 l tid
-
7/30/2019 Day 3 - Transcription and RNA Processing
44/50
16S rRNA
1. ~1500 nucleotids
2. Many modified bases
3. Compact 3-D folding4. Complexed with 21
proteins
5. Highly conserved
RNA i
-
7/30/2019 Day 3 - Transcription and RNA Processing
45/50
Group I introns
Group II introns
Introns in eukaryotic mRNAs
RNA processingIntrons and splicing
Splicing: Removal of parasiticDNA information from RNA
Box 2.6
-
7/30/2019 Day 3 - Transcription and RNA Processing
46/50
Removal of
parasitic DNAinformation fromproteins
Removal of inteins
GyrA of many prokaryotes
contains an intein
Box 2.6
VMA1 protein of yeast
Intein
1 284 738 1071
N-extein C- extein
454
RNA Editing: Edited ND1 mRNA of T brucei * deleted U
-
7/30/2019 Day 3 - Transcription and RNA Processing
47/50
uGAUACAAAAAAACAUGACUACAUGAUAAGUAuCAuuuuAuGuuAuuuuuGGuAGuuuuuuuACAuu
uGuAuCGuuuuACAuuuG*GUCCACAGCAuCCCG***CAGCACAuG**GuGuuuuAuGuuGuuuAuuGuA
uuuuuGuGGuGA*AuuuAuuGuuuA**UAUUGAuUGuAuuAuA***G*GuuAUUUGCAUCGUGGUACAG
AAAAGUUAUGUGAAUAUAAAAGUGUAGAACAAUGUCUUCCGuAUUUCGACAGGUUAGAuuAuG
uuA*GuGuuuGuuGuAAuGAGCAuuuGuuGuCuuuA***UGuuuuGAGuAuAuGuuGCGAuGuuGuuuGu
CGuuACGuuGuGCAuuuAuGCGuuuAuuAAuuGuA****GAAuuuAC***CCGuAGuuuuAAuGGuuuGuuGuGuAuAuCAuGuAuGGuuuuGG*AuuuAGGuuGuuuGuCUCCGuuG*UUAuGAuCAuuuGAGGAA***
CG*UGACAAAuuGAuGACAuuuuuuGAuuuAuG**UUGuGGuuGuCGuAuGCAuuuGGCUUUCAuGGu
uuuAuuA*GGuAUUCUUGAUGAuuuuGuuuuuGGuuuuGuuGAuuuuuuGuuGuuGuuGA***UAAuAuC
AuGuuuGuuuGuuAuGGAuuGuuAuGAuuuGuuAuuuGuGGGuAAUCGuuuAuuuUAuuuGCGuuuGC***GuGGuuuGuCAuuuuuuGAuuuAuAuGAuuuA**GuuuuuA**A**UAGuuuAAGuGGuGuuuuGuCuCGu
uCGuuAGGuAuGGuGuGAGAuuGUCGuuuAuuuAGuuGuuA****UGA*****GuUGuAuuuuAuGuuuuG
uuAuGAuuAuuGuuuuuGuuuuAuAGGuGAuGCAuuuGA*UCGuuuAuuuuuACGuuuGuuuGAUAuGC
GuAuGAGuuuGuuGAuuuGuAAGCAAuGuuuuuuuGuuGGuuuuuuuGuuuuuG*****GuuuuGuuuGuuuGuuuG**AuuAuuuAuAuuGuGAuAuuACCAuuG****AGACCAuuAuuAuGuuAuuuuAuAGuuuGuGGu
GuuGuuGuuuGCCGGGuAuA*UCAuuuGC*UUGUGuuGAACACCCCAAAGGuGA***GuAuuGuuuGu
uAuuA****UGuuuuuGuGuuGGuuuAuGuuCUCGuuuACGuuuGCGuuGuGCGGAuuuuuuGCA*UAUU
UGuuuAuuGGAuGuuuGuuuGCGuGGuuuuuuAuuGCAuGAuuuAGuuGC***C*GuuuuAGGuAAuAuuGAuGuuGuuuuuGGAuCCGUAGAUCGuuA*GuuuuAuAuGuG**A******GGUUAUUGuAGGAUUGUU
UAAAAUUGAAUAAAAA
RNA Editing: Edited ND1 mRNA ofT. bruceiu added U
Courtesy of Dr. Donna Koslowsky
Mitochondrial RNA Editing in Trypanosomes
-
7/30/2019 Day 3 - Transcription and RNA Processing
48/50
Mitochondrial RNA Editing in Trypanosomes
Editing a substrate RNA by an editosome using a guide RNA
Substrate mRNAs are transcribed from mtDNA maxicircles (5-6)Guide RNAs are transcribed from mtDNA minicircles (~1000)
Modified from Catteneo (1990)
AUAUAAAAGCGGGAGUUAUUUUUAUUAUUUUUU 3
UAAAAGUAAUAAA5
A
G CC
C
A
C
C
C
A
AAAA
G
U
UUUUUUUUU
U
UU UU
* .... .. .... ..
5
3
AnchorTether
GUIDE
5
ATATAAAAGCGGGAGTTA A A 3DNA
Transcript
Guide RNA
Edited segment
EDITOSOME
-
7/30/2019 Day 3 - Transcription and RNA Processing
49/50
Mitochondrial and Chloroplast RNAs
Untranslated regions (UTRs) and secondary structuremodifications of nuclear RNAs in eukaryotes
RNA Editing: Base Modifications
C U Editing in the co 2 mRNA of mai e mitochond ia
-
7/30/2019 Day 3 - Transcription and RNA Processing
50/50
C U Editing in the cox2 mRNA of maize mitochondria