Biochemistry 304 2014 Student Edition TRANSCRIPTION

Student Edition
6/3/13 Version
Dr. Brad Chazotte
213 Maddox Hall
[email protected]
Web Site:
Original material only ©2007-14 B. Chazotte
Pharm. 304
Fall 2014
•Examine the different types of RNAs
•Examine the mechanism of RNA replication.
•Learn what steps are involved in transcribing DNA to RNA.
•Examine which enzymes/proteins are involved in transcription.
•Understand how RNA are modified post-transcriptionally
•Understand how RNAi’s are involved in post-transcription
•Examine the similarities and differences between prokaryotic and
eukaryotic transcription.
mRNA (messenger) – basic idea: the sequence of its nucleotides derived
from a DNA sequence (gene) is converted (transcribed) by the ribosomes
into a protein (sequence of amino acids).
rRNA (ribosomal) – forms part of the structure, ribosome, that synthesizes
tRNA (transfer) - small compact molecule that delivers specific amino
acids to ribosome for protein synthesis.
RNAi (interference) a class of small non coding RNAs that function in post
transcription regulation as a silencing mechanism
Long Noncoding RNA (lncRNA) extensively transcribed RNAs that do
NOT code for proteins that form extensive networks of ribonucleoprotein
complexes (RNPs) with numerous chromatin regulators that then target
these enzymatic activities to appropriate locations in the genome
RNA polymerase (RNAP) - enzyme that synthesizes RNA
Selected Noncoding RNAs
Voet, Voet & Pratt 2012 Table 26.1
The process by which a sequence of DNA nucleotides (gene) are
copied (transcribed) to RNA.
Major difference between Prokaryotes and Eukaryotes:
Prokaryote (single cell organism) – almost all the DNA is
Eukaryote (multicellular & has nucleus) – most of the
DNA is not transcribed. Therefore, control
mechanisms need to regulate what is transcribed,
e.g., consider liver vs brain cell’s structure/function
and proteins
Sequence for the Transcription Process
Initiation, Elongation, & Termination
•Find where to start – promoter sequence on DNA that RNA
polymerase recognizes.
•Get the DNA set – DNA strands must be temporarily unwound
so RNA polymerase can read DNA.
•Copy the DNA→RNA – RNA polymerase reads the DNA and
synthesizes RNA.
•Know when to stop - RNA polymerase comes to termination
site (sequence).
•Goodbye - RNA separates from the RNA polymerase.
•Modify RNA – some RNAs may undergo posttranscriptional
Function: Synthesize RNA based on DNA template.
Structure: Negative charges uniformly over enzyme outer surface,
but inner walls of channel are positively charged. WHY?
One enzyme synthesizes all RNAs
Four to five different enzymes each synthesizing a
different class of RNA
Berg, Tymczko, & Stryer 2012 Fig 29.1
Prokaryote: E. Coli
Holoenzyme: 2’σ “whole enzyme”, binds loosely to
DNA but specifically to promoter sites.
Core enzyme: 2’ Carries out RNA polymerization. It
binds tightly but NON-SPECIFICALLY to DNA
The sigma subunit, σ, helps the enzyme to bind
loosely to DNA but specifically to promoter regions on
DNA. Dissociates after initiation.
Berg, Tymcko, & Stryer 2012 Table 29.1
Sense and Antisense DNA Strands
Only one of two DNA strands transcribed by
RNA Polymerase to make RNA strand
Antisense (noncoding) strand: the DNA strand that is the
template for RNA – it is complementary to the RNA
Sense (coding) strand: the DNA strand that has the same
sequence as the RNA differing only in thymine for RNA’s
Voet, Voet & Pratt 2013 Fig 26.4
Lehninger (Nelson & Cox) 2005 Fig 26.3
Prokaryotes may have multiple genes
controlled together
E. Coli lac operon
Prokaryotes: (above illustration) genes are frequently
arranged in tandem so that they can be transcribed
together. Lac operon codes 3 proteins (enzymes) for
lactose metabolism. The separate regulatory gene codes
for a repressor protein that inhibits lac operon
Eukaryotes: are different – most genes coding proteinsare individually transcribed
Voet, Voet & Pratt 2013 Fig 26.5
RNAP Binds to Promoters for Initiation
Sense Strand Sequences of selected E. Coli Promoters
TATA box
Promoter: sequence on 5’ side of initiation site to which RNAP holoenzyme tightly
binds. One is TATA box ~ -10 upstream of initiation.
Important: The rate of gene transcription depends on the rates at which promoter
forms a stable initiation complex with RNAP holoenzyme. The closer to consensus
sequence the stronger the promoter.
Consensus sequence: an “average” sequence found for many promoters
Voet, Voet & Pratt 2013 Fig 26.6
Different Promoter Sequences & Sigmas
Bacteria have different sigmas that recognize different promoters.
Determines which genes are transcribed
Sigma recognizes two sequences – give greater specificity, more
precise control, less chance of error ( a good thing). Second
centered ~ -35
Berg, Tymoczko & Stryer 2012 Fig. 29.11
Initiation &
Elongation by
E. Coli RNA
Multiple transcriptions can
occur along DNA molecule
RNAP is processive – once the
open complex is formed the
enzyme proceeds along the
template without dissociating.
Voet, Voet & Pratt 2013 Fig 26.9
Lehninger (Nelson & Cox) 2005 Fig 26.6
DNA Supercoiling during
~17 bp
Voet, Voet & Pratt 2013Fig 26.8
Transcription Frequency
•Transcription frequency is different for different genes
Constitutive Enzymes – synthesized at a ~ constant rate.
Typically involved in basic cellular functions
Inducible Enzymes – synthesis depends on cell’s needs.
•Gene expression is found to be significantly controlled via
mechanisms that regulate the rate of transcription.
•Control can also result from the stability of transcription
products. mRNA are much shorter lived than rRNA. Why?
Termination of Transcription
In E. Coli many gene transcription (spontaneous) termination
sequences have:
•AT base pairs – series of 4-10 consecutive bases pairs with A’s on
template strand. Termination in or just past this sequence.
Voet, Voet & Pratt 2008 Fig 26.9a
•G+C-rich region with a palindromic sequence
immediately before the series of A-T’s.
Voet, Voet & Pratt 2008 Fig 26.9b
Rho Factor Terminates Transcription
A protein hexamer; a helicase catalyzing
the unwinding of DNA-RNA or RNA-RNA
double helices.
•Induces termination of non-spontaneous
terminating sequences
•Enhances efficiency of spontaneously
terminating sequences.
Rho binds to an RNA recognition site and
slide in 5’3’ direction till encountering
RNAP paused at termination site. Rho
unwinds DNA-RNA at transcription bubble
which releases the RNA.
(Rho-RNA Complex)
Voet, Voet & Pratt 2008 Fig 26.10
Eukaroytic Transcription
Some key differences with prokaryotes:
Multiple RNA polymerases (differ in RNAs they synthesize)
Relatively complex control sequences
More complex process involving > 100 polypeptides
In eukaryotes transcription
and translation are separated
in space. Transcription of the
genome occurs in the
Berg, Tymoczko & Stryer 2012 Fig. 29.21
Eukaryotic RNA Polymerases
•RNAP I - synthesizes most rRNA precursors, located in nucleoli
•RNAP II - synthesizes mRNA precursors, located in nucleoplasm
•RNAP III - synthesizes 5S rRNA precursors, tRNAs & variety of
other small nuclear and cytosolic RNAs, located in
Do not have a σ factor like
prokaryotes, but the RNAP is
recruited to the initiation site by
accessory proteins that recognize
promoters. Each RNAP recognizes a
different promoter.
Yeast RNA Polymerase II Structure
Voet, Voet & Pratt 2013 Fig 25.12
RNA Polymerase II RNA Elongation
Voet, Voet & Pratt 2013 Fig 26.13b
Eukaroytic Promoters
Some key differences with prokaryotes:
Each RNA Polymerase has it’s own promoters.
Eukaryotes also have ENHANCERS: DNA sequences
that can enormously increase the effectiveness of
promoters. Play a key role in gene expression in a
specific tissue or during development. Position on DNA
relative to promoters is NOT fixed.
Transcription factors (coded by gene elsewhere) enable
RNA polymerase to find it’s specific initiation site, e.g.
Class I TF for RNAP I, Class II for RNAP II .
Cis-acting : control elements on the same DNA strand.
Trans-acting: control elements from a gene on a different DNA
strand, e.g. transcription factors.
Eukaryotic Genes Promoter
CCAAT box located upstream of many structural genes
Constituently transcribed genes expressed in all tissues
have the GC box upstream of transcription start site
Structural genes selectively transcribed in one
or a few cell types lack GC box but have ATrich sequence resembling the TATA box
Eukaryotic RNA polymerases do
NOT bind to actual promoter.
Berg, Tymoczko & Stryer 2012 Fig. 29.23
Berg, Tymoczko & Stryer 2012 Fig. 29.24
Voet, Voet & Pratt 2013 Fig 26.15
Voet, Voet & Pratt 2006 Fig 25.13
Eukaryotic General Transcription Factors
Similar to the function of prokaryotic σ-factor’s function,
eukaryotes utilize a complex of six or more general
transcription factors to bind to the promoter region and
initiate transcription.
TFs, polymerase, and promoter DNA combine to form a
Voet, Voet & Pratt 2013 Table 26.2
PIC Assembly on a TATA Box Promoter
Protein-protein and protein-DNA
interactions by transcription factors
alter conformations causing changes
in structure/function.
Note: TF induced kink in DNA
(white molecule)
Voet, Voet & Pratt 2013 Fig 26.19
Voet, Voet & Pratt 2013 Fig 26.17
Transcription Inhibition &
Many antibiotics are highly specific inhibitors of
biological processes.
A wide variety of compounds can inhibit
transcription in prokaryotes and eukaryotes.
Rifamycin B specifically inhibits prokaryotic but not
eukaryotic transcription by preventing elongation.
Actinomycin D tightly binds to double helical DNA
and prevents it from being an effective template for
Actinomycin D phenoxazone ring
intercalates between DNA base pairs
unwinding the DNA 23˚and separating
neighboring base pairs 7Å
Voet, Voet, & Pratt 2013 Box 26.2
Posttranscriptional Processing
Primary transcript: the immediate product of transcription
RNAs are modified in a number of ways after transcription:
Appending of nucleotide sequences to 3’ & 5’ ends
Exo and endonucleolytic removal of polynucleotide
Modification of specific nucleotide residues
mRNA Processing
Most mRNAs undergo no modification after
mRNAs are transported out of the nucleus where they
are synthesized - can undergo extensive
posttranscription processing before leaving the
Eukaryotic 5’ Cap & Poly(A) Tail on mRNA
Cap protects
mRNA from
residue joined via a 5’5’ bridge to transcript’s
initial 5’nucleotide
Voet, Voet & Pratt 2013 Fig 26.20
Poly(A) tail
thought to protect
mRNA from
Mature mRNAs have ~250 nt Poly(A) tail, degradation. The
i.e., on their 3’ end.
older the mRNA
the shorter the
Berg, Tymoczko & Stryer 2012 Fig. 29.31
Eukaryotic Genes Contain Exons & Introns
Primary Transcripts must be Edited
Introns: Nonexpressed intervening sequences are excised
from pre-mRNAs
Exons: Expressed sequences of mRNA. Edited (spliced )
together after intron excisions.
Production of mature mRNAs
Voet, Voet & Pratt 2013 Fig 26.22
How are Eukaryotic Exon-Intron
Junctions Marked in mRNAs?
•High sequence homology at exon-intron boundary – note
percentages next to base symbols.
•The 3’ splice is preceded by a sequence of 11 predominately
pyrimidine nucleotides.
•Invariant GU at intron 5’ boundary
•Invariant AG at intron 3’ boundary
Consensus Sequences at Exon-Intron Junction
Voet, Voet & Pratt 2013 Fig 26.23
Eukaryotic mRNA Splicing
Two transesterification reactions involved:
1. Form LARIAT structure from 2’5’ phosphodiester bond
between intron’s 5’ terminal phosphate & intron
adenosine residue (20-50 residues upstream of 3’ end).
The 5’ exon’s free 3’OH
displaces 3’ end of intron to
form a phosphodiester bond
with 5’ terminal phosphate of
Voet, Voet & Pratt 2013 Fig 26.24
Spliceosome and snRNAs (“snurps”)
Spliceosome: 45S particle where splicing occurs. Forms
complex with pre-mRNA, snRNPs, & pre-mRNA binding
•Preassembled spliceosome binds to pre-mRNA
•Spliceosome undergoes conformational changes as it carriers
out the two esterification reactions to excise intron and join
adjacent exons.
Structure of U1-snRNP
small nuclear riboproteins
U1-snRNP recognizes complementary
consensus sequence at 5’ intron boundary
Lehninger (Nelson & Cox) 2005 Fig 26.16c
Berg, Tymoczko & Stryer 2012 p. 876
Voet, Voet & Pratt 2008 Fig 25.25b
What are the Advantages of Gene Splicing?
•Can promote rapid evolution of proteins
Noted that many eukaryotic proteins have a modular design. Likely that
the genes encoding these modular proteins have arisen via the stepwise collection of
exons assembled by recombination between their neighboring introns.
•Can provide a means from a single gene to encode multiple
proteins via Alternative Splicing.
The expression of numerous cellular genes can be modulated by the
selection of alternative splice sites. As such one may have exons in one cell type be
introns in another cell type. Gives greater protein diversity in fewer completely
separate genes.
Alternative Splicing
•Occurs in all multicellular organisms.
•Especially prevalent in vertebrates
•Some 60% of human structural genes are subject to it.
Exons can be retained or skipped
Alternative Splice Site Selection in
Drosophila Sex Determination Pathway
Introns may be excised or retained
5’ or 3’ splice sites can be shifted to
make exons longer or shorter
Transcription start site or
polyadenylation site can be altered to
further diversify of a gene’s product
Voet, Voet & Pratt 2006 Fig 25.26
mRNA Stability/Half-Life
•Most mRNAs have limited stability
•In Eukaryotes half-life ~ 30min or less
•Shortest half-life for coding for regulatory proteins
•Long sequence of AU-rich nucleotides can code for
enhanced degradation by stimulating removal of
poly(A) tail.
•(Compared to Prokaryotes typical half-life ~ 3 min.)
ALberts, Bray et al 1994 p 464
RNAi – Post-transcriptional
Regulation of mRNA
(Gene Silencing)
Roles of Small ncRNAs in Cells
Group 1: the siRNAs - target
mRNAs for destruction
Group 2: the miRNAs – generally
regulate protein translation from
Group 3: the short siRNAs which
target chromatin for modification
Elliot & Ladomery “Molecular Biology of RNA” 2011 Table 18.1
Important RNAi Definitions
Ribonucleic Acid interference (see Fire et al 1998)
•siRNA Short interfering RNA. These are dsRNA 21-25bp in length
with a 3’ overhang that are processed from longer RNAs by the
enzyme “Dicer”. Synthetic siRNA can be introduced in mammalian
cells to produce interference.
•miRNA microRNA. ssRNA 19-23nt long that originate from ss
precursor transcripts characterized by imperfectly base-paired hairpins.
Function as a silencing complex.
•piRNA PIWI-interacting RNAs 24 -32 nt long involved in germ line
cells. Function: Block transposons
•shRNA Short (interfering) hairpin RNA. Used to supply siRNAs
with vector-based approaches to produce stable gene silencing ( a
research, experimental approach to add exogenous RNAs)
•RISC RNA-induced silencing complex. A nuclease complex of
proteins and siRNA, miRNA, etc. that targets and cleaves mRNAs
complementary to the siRNA, miRNA, in the RISCRNAi
& Epigentics Sourcebook, Invitrogen 2010
How RNA interference (RNAi) works
This illustration is
based on in vitro
approaches to using
RNAi in research
RNAi & Epigentics Sourcebook, Invitrogen 2010
In this process the antisense strand of the siRNA
duplex becomes part of a multi-protein complex, or RNAinduced silencing complex (RISC),
RISC identifies the corresponding mRNA and
cleaves it at a specific site.
This cleaved message is targeted for degradation -results in the loss of protein expression.”
Can induce RNAi as:
• synthetic molecules
• RNAi vectors
In vitro dicing of RNA (Figureleft). In mammalian cells, short
pieces of dsRNA, short
interfering RNA (siRNA),
initiate the specific degradation
of a targeted cellular mRNA.
RNAi & Epigentics Sourcebook, Invitrogen 2010
•Are small noncoding RNAs that play important roles in
posttranscriptional gene regulation.
•May comprise a new layer of regulatory control over gene
expression programs in many organisms.
•In animal cells miRNAs regulate their targets by
translational inhibition and mRNA destabilization
Ann Rev Cell Biol 23 175-205 2007
Naturally occurring and evolutionarily conserved
Effect gene expression
Most miRNAs transcribed by RNA Polymerase II –form
stem-loop structures
Located either within mammalian introns or exons of
protein-coding genes (70%) or intergenic areas (30%).
Found to downregulate gene expression by base-pairing
with the 3’ untranslated regions (3’UTRs) of target
Annu Rev Cell Biol 23 175-205 2007
Annu Rev Med 60 167 2009
miRNA Biogenesis
Pri - Primary transcript
•miRNA gene transcribed by Pol
•Drosha an RNase III
endounclease & DGR8/Pasha
protein in nucleus cleave PrimiRNA to give 2nt overhang
•Exportin-5 transports pre-miRNA
into cytoplasm
•Cleaved by the Rnase III
endonuclease, Dicer, and
TRBP/loquacious protein
•Release 2-nt 3 overhang
containing 21nt
•miRNA strand loaded into an
Argonaute containing RISC
•(miRNA* typically degraded)
Annu Rev Cell Biol 23 175-205 2007 Fig. 1
Differ from miRNAs mainly in their origin.
siRNA derive from endogenous or exogenous dsRNAs and are
processed into smaller siRNAs by Dicer
siRNAs usually induce cleavage of their targets when loaded
onto an Ago2-containing RISC (RNA-induced silencing
Can also act like miRNAs on targets with imperfect
complementarity and induce translational repression
Ann Rev Cell Biol 23 175-205 2007
•Small non-coding RNAs involved in gamateogenesis
(Mammals generate huge numbers of pachytene piRNAs {pachytene a stage in meiosis}
that do not match transposon sequence – function currently unknown)
•Interact with piwi proteins and Argonaute proteins.
•Contain partial and complete (complementary) transposon
sequences that serve as memory banks for the pi system
analogous to how the immune system defends an organism.
(Think of it as a “genome immune system”.
•piRNAs track down a transposon and Piwi proteins slice up
the rogue DNA.
•Some scientists postulate that they adjust gene expression
viz. siRNAs and miRNAs.
•piRNAs have recently been found in central nervous system
– maybe involved in memory
[Thompson & Liu Annu. Rev. Cell Dev. Biol. 25:355-376 2009
Leslie Science 339:25-27 2013]
RNA-Induced Silencing Complex
•Complex with an RNAi, Argonaute protein and other
•Uses the RNAi (miRNA, siRNA, etc.) to target a
mRNA in the 3’ UTR region and bind to it.
•The complex proceeds to degrade the mRNA so the
gene product is not expressed.
RNAi in Research
“RNAi is a specific, potent, and highly successful approach
for loss-of-function studies in virtually all eukaryotic
Several appropriate tools to induce RNAi:
•Chemically synthesized siRNA and shRNA
•miR RNAi-encoding plasmid and viral vectors
Select one of the above depending on the model system, the
length of time required for knockdown, and other
experimental parameters
Invitrogen web site need reference
miRNAs in Cancer, Viral
Infection, & Disease
•CANCERS miRNA levels are altered in primary human tumors.
Loss of miRNAs in cancer tissues may suggest a role for miRNAs
as tumor suppressors.
•Significantly different miRNA profiles can be assigned to various
tumor types – may be of diagnostic use.
•VIRUS INFECTION Viruses use miRNAs in their effort to
control their host cell, while reciprocally host cells use miRNAs
to target essential viral functions.
•e.g. Tourette’s Syndrome miRNA may be involved due to a
mutation. Replacement of a GU pair with UA pairing in 3’UTR
region of SLITRK1 causes stronger miRNA regulation.
Ann Rev Cell Biol 23 175-205 2007
Long Noncoding RNAs (lncRNA)
Are key regulatory layers in global gene expression
There are now numerous examples of lncRNAs in
controlling access or dismissal of regulatory proteins
from chromatin. Epigenetics - lncRNAs affect packing
of DNA – which, in turn, affects gene expression.
Common emerging theme: “they form ribonucleic acid-protein
interactions to carry our their functions by modulating chromatinmodifying complexes, by interactions with transcription factors,
and likely by many additional mechanisms.”
Enhancers transcribe RNA. “Two classes of enhancer RNAs have
been identified that are by-products of transcription and lncRNAs
that play a role in forming enhancer contacts to promote gene
Rinn & Chang Annu. Rev. Biochem 81:145-166 2012
Long Noncoding RNAs:
Structure & Function
lncRNAs adopt 2° and 3° structures that relate to function. Being able to
bind to protein partners facilitates their regulatory capacities in gene
expression (facilitating or silencing).
•Decoys: At simplest level lncRNAs can serves as decoys that preclude
access of regulatory proteins to DNA, i.e. prevent DNA-biding proteins
from binding to DNA
•Scaffold: lncRNAs can serves as adaptors to bring two or more proteins
into discrete complexes. Hundreds of lncRNAs have been identified that
form ribonucleic-protein interactions with multiple protein partners.
•Guides: Many lncRNAs are individually required for the proper
localization of specific protein complexes. Can serve to target gene
silencing in an allele-specific fashion. These lncRNAs combine two basic
molecular functions – (a) binding a protein partner, (b) a mechanism to
interface with selective regions of the genome.
Rinn & Chang Annu. Rev. Biochem 81:145-166 2012
lncRNAs & Disease
lncRNAs have emerged as key role-players in the
etiology of several disease states.
•Cancer: Dozens of lncRNAs have been documented to have
altered expression in human cancers and are regulated by
specific oncogenic and tumor suppression pathways, e.g.
P53, MYC and NF-κB
•Other: Currently hypothesized that lncRNAs are involved in
the pathogenesis of many other diseases.
Hundreds of genomic regions that do not contain proteincoding genes are strongly associated with a wide spectrum of
human diseases.
Rinn & Chang Annu. Rev. Biochem 81:145-166 2012
rRNA Processing
In E. Coli, e.g., 5S, 16S, & 23 S rRNA on a primary
polycistronic transcript that undergo processing.
Genes transcribed and processed in the nucleolus.
Primary transcript includes 18S, 5.8S & 28S rRNA.
Some eukaryotic rRNAs are self-splicing, i.e. act as
own enzyme!
Posttranscriptional Processing of
E. Coli rRNA
Primary Processing : Separate polycistronic primary transcript into separate
rRNAs (and 4 tRNAs).
Secondary Processing: Trim 5’ & 3’ ends of pre-rRNAs. Methylation
(for protection?) of specific residues during ribosome
Voet, Voet & Pratt 2013 Fig 26.29
Eukaryotic rRNA Processing
Primary 45S transcript is methylated then cleaved
into 5.8S, 18S & 28S.
Self-Splicing of
Tetrahymena pre-rRNA
Methylation sites targeted by small nucleolar
(snoRNA) encoded by introns(!) of structural
genes. Methyltransferase enzyme does
Some rRNAs (group I introns) are self-splicing
carrying out transesterification reactions. Occur in
nuclei, mitochondria, and chloroplasts, but NOT
Voet, Voet & Pratt 2013Fig 26.30
tRNA Processing
•tRNAs are initially made longer and processing shortens them.
•RNase P cleaves 5’ and RNase D cleaves 3’ ends
•CCA added to 3’ end of all tRNAs
•Specific bases are modified (methylation, deamination, or reduction)
(some eukaryotic tRNAs have introns that must be excised)
Lehninger (Nelson & Cox) 2005 Fig 26.23
Lehninger (Nelson & Cox) 2005 Fig 26.24
End of Lectures

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