Review from last time
• Coding and non-coding repeats make up the moderately
repetitive portion of the eukaryotic genome
• Polyploidization and gene duplication contribute to
structural and functional genome change
• Transposable elements contribute to structural and
functional genome change
• Comparative genomics is a useful technique to
determine the conserved (likely functional) portions of a
genome
Chapter 11:
Gene Expression:
From Transcription to Translation
details
This Chapter in One Slide
details
details details
details
Gene Expression
• RNA – Ribonucleic acid
– Slightly different from DNA
– Uracil instead of Thymine
• RNA is critical to all gene expression
• mRNA – messenger RNA; created from a
DNA template during transcription
• tRNA – transfer RNA; carriers of amino
acids; utilized during translation
• rRNA – ribosomal RNA; the site of
translation
• Other RNAs – snoRNA, snRNA, miRNA,
siRNA
• Many RNAs fold into complex secondary
structures
Transcription
• Transcription – the process of copying a DNA template
into an RNA strand
• Accomplished via DNA dependent RNA polymerase (aka
RNA polymerase)
Transcription
• By the end of this series, you should be able to explain
much of this animation
• http://www.as.wvu.edu/~dray/219files/Transcription_588x392.swf
• Bacterial RNA polymerase 50 - 100
nucleotides/sec
• Most genes are transcribed simultaneously by
numerous polymerases
• Polymerase moves along DNA in 3' —> 5' direction
• Complementary RNA constructed in ____ direction
• RNAn + NPPP —> RNAn+1 + PPi
Transcription
• How does the polymerase know where to start?
• Promoter = the assembly point for the transcription
complex
• RNA polymerases cannot recognize promoters on their
own - transcription factors
• Transcription factors - enzymes have evolved to
recognize (physically interact with) specific DNA
sequences and with other proteins
• The promoter is one such DNA sequence
Transcription
• Prokaryotic Transcription
• Similar DNA sequences are seen associated with genes in roughly
the same location for multiple genes in bacteria
– The consensus sequence is the most common version of such a
conserved DNA sequence
TTGATA
TTGACA
CTGACG
– DNA sequences just upstream from a large number of bacterial genes
have 2 short stretches of DNA that are similar from one gene to another
(-35 region & -10 region)
• T78T82G68A58C52A54 -- 162117521819 -- T82A89T52A59A49T89
•
- 35 region
spacer
-10 region
Transcription
• Prokaryotic Transcription
• Bacterial promoters are located just upstream of the
RNA synthesis initiation site
– The nucleotide at which transcription is initiated is called +1; the
preceding nucleotide is –1
– DNA preceding initiation site (toward template 3' end) are said to
be upstream
– DNA succeeding initiation site (toward template 5' end) are said
to be downstream
Transcription
• Prokaryotic Transcription
• One RNA polymerase with 5
subunits tightly associated to
form core enzyme
• Core enzyme minus sigma (σ)
factor will bind to any DNA.
– By adding σ, RNA pol will bind
specifically to promoters (-10 & 35 sequences)
Transcription
• Eukaryotic vs. Prokaryotic
Transcription
• Much of what we know is derived from
studies of RNA pol II from yeast
– 1. Seven more subunits than its bacterial
RNA pol
– 2. The core structure & the basic
mechanism of transcription are virtually
identical
– 3. Additional subunits of eukaryotic
polymerases are thought to play roles in
the interaction with other proteins
– 4. Eukaryotes require a large variety of
accessory proteins or transcription factors
(TFs)
Review from last time
• Basic ideas behind transcription and translation
• To get from DNA to functional protein, many types of
RNA are critical
• RNA differs chemically from DNA in only two ways
• RNA tends to form secondary structures
• RNA polymerase initiates transcription at promoter sites
with the aid of transcription factors like sigma (in
prokaryotes)
• Promoters are DNA sequences that act to direct RNA
polymerases to the appropriate position
Transcription
• Eukaryotic Transcription - one major difference
• Three distinct RNA polymerases, each responsible for
synthesizing a different group of RNAs
– RNA polymerase I (RNA pol I) - synthesizes the larger rRNAs
(28S, 18S, 5.8S)
– RNA polymerase II (RNA pol II)- synthesizes mRNAs & most
small nuclear RNAs (snRNAs & snoRNAs)
– RNA polymerase III (RNA pol III) - synthesizes various small
RNAs (tRNAs, 5S rRNA & U6 snRNA)
Transcription
• Eukaryotic Transcription - RNA processing
• All major RNA types (mRNA, tRNA, rRNA) must be processed
– Terminology
– The primary (1°) transcript is equivalent in length to the DNA
transcribed
– The corresponding segment of DNA from which 1° transcript is
transcribed is called transcription unit
– The 1° transcript is short-lived; it is processed into smaller,
functional RNAs
– Processing requires variety of small RNAs (90 – 300 nucleotides
long) & their associated proteins
Transcription – mRNA
• Messenger RNAs (mRNA)
• Transcribed by RNA pol II
• Remember this?
• http://www.as.wvu.edu/~dray/219files/Transcription_588x392.swf
• Polymerase II promoters lie to 5' side of each
transcription unit
– In most cases, between 24 & 32 bases upstream from
transcription initiation site is a critical site
– Consensus sequence that is either identical or very similar to
5'-TATAAA-3‘, the TATA box
– The site of assembly of a preinitiation complex
• contains the GTFs & the polymerase
• must assemble before transcription can be initiated
Transcription – mRNA
• The preinitiation complex
• Step 1 - binding of TATA-binding
protein (TBP)
– Purified eukaryotic polymerase, cannot
recognize a promoter directly & cannot
initiate accurate transcription on its own
– TBP is part of a much larger protein
complex called TFIID
– TBP kinks DNA and unwinds ~1/3 turn
Transcription – mRNA
• The preinitiation complex
• Step 2 – Binding of ~8 TAFs (TBPassociated factors) to make up the
complete TFIID complex
• Step 3 – Binding of TFIIA (stabilizes
TBP-DNA interaction) and TFIIB
(involved in recruiting other TFs and
RNA pol II)
Transcription – mRNA
• The preinitiation complex
• Step 4 – RNA pol II and TFIIF bind via
recruitment by TFIIB
• Step 5 – TFIIE and TFIIH bind
• TFIIH is the key to activating
transcription in most cases
• TFIIH is a protein kinase –
phosphorylates proteins
• TFIIH may also act as a helicase
Transcription – mRNA
• The preinitiation complex
• All these general transcription factors and pol II are enough to
generate basal transcription
• Transcription can be upregulated or downregulated by a huge
diversity of other cis and trans acting factors to be discussed in
chapter 12.
• Once an mRNA is produced, it must be processed.
• Processing involves the addition of a cap, the addition of a poly-A
tail, and splicing out of introns.
RNA processing – mRNA
• Transcription generates
messenger RNA
– A continuous sequence of nucleotides
encoding a polypeptide
– Transported to cytoplasm from the
nucleus
– Attached to ribosomes for translation
– Are processed to remove noncoding
segments
– Are modified to protect from
degradation and regulate polypeptide
production
RNA processing – mRNA
• RNA polymerase II assembles a 1° transcript that is
complementary to the DNA of the entire transcription unit
• 1° transcript contains both coding (specify amino acids)
and noncoding sequences
• Subject to rapid degradation in its raw state
RNA processing – mRNA
• 5’ cap
• 5' methylguanosine cap forms very
soon after RNA synthesis begins
– 1. The last of the three phosphates is
removed by an enzyme
– 2. GMP is added in inverted
orientation so guanosine 5' end
faces 5' end of RNA chain
– 3. Guanosine is methylated at
position 7 on guanine base while
nucleotide on triphosphate bridge
internal side is methylated at ribose
2' position (methylguanosine cap)
RNA processing – mRNA
• 5’ cap
• Possible/known functions of 5’
cap
– May prevent exonuclease digestion
of mRNA 5' end,
– Aids in transport of mRNA out of
nucleus
– Important role in initiation of mRNA
translation
RNA processing – mRNA
• Polyadenlyation
• The poly(A) tail – 3' end of most mRNAs contain a string
of adenosine residues (100-250) that forms a tail
– Protects the mRNA from degradation
– AAUAAA signal ~20 nt upstream from poly(A) addition site
– Poly(A) polymerase, poly(A) binding proteins, and several
cleavage factors are involved
– http://www.as.wvu.edu/~dray/219files/mRNAProcessingAdvanced.wmv
RNA processing – mRNA
• mRNA processing – Splicing
• Requires break at 5' & 3' intron ends (splice sites) &
covalent joining of adjacent exons (ligation)
• http://www.as.wvu.edu/~dray/219files/mRNASplicingAdvanced.
wmv
• Why introns?
– Disadvantages – extra DNA, extra energy needed for processing, extra
energy needed for replication
– Advantages – modular design allows for greater variation and relatively
easy introduction of that variation
RNA processing – mRNA
• mRNA processing – Splicing
• Splicing MUST be absolutely precise
• Most common conserved sequence at eukaryotic exonintron borders in mammalian pre-mRNA is G/GU at 5'
intron end (5' splice site) & AG/G at 3' end (3' splice site)
RNA processing – mRNA
• mRNA processing – Splicing
• Sequences adjacent to introns contain preferred
nucleotides that play an important role in splice site
recognition
Review from last time
• Transcription cannot proceed until the pre-initiation
complex has been constructed at the promoter
• Construction of the pre-initiation complex is a stepwise
recruitment process that eventually brings in RNA pol II
• Multiple transcription factors are involved, know them
and their functions
• The primary transcript is capped almost immediately by
a methylguanosine nucleotide that serves multiple
functions
• The 3’ end of the transcript is cleaved and a poly-A tail is
added
• Splicing of the primary transcript must be precise and
multiple sequence-based landmarks aid the process
RNA processing – mRNA
• mRNA processing – Splicing
• Nuclear pre-mRNA (common)
– snRNAs + associated proteins =
snRNPs
• snRNAs – 100-300 bp
• U1, U2, U4, U5, U6
– 3 functions for snRNPs
• Recognize sites (splice site and
branch point site)
• Bring these sites together
• Catalyze cleavage reactions
– Splicosome – the set of 5 snRNPs
and other associated proteins
– Summary movie available at:
– http://www.as.wvu.edu/~dray/219fil
es/mRNAsplicing.swf
RNA processing – mRNA
• mRNA processing –
Splicing
• 1. U1 and U2 snRNPs bind
via complementary RNA
sequences
• Note the A bulge produced
by U2
• U2 is recruited by proteins
associated with an exon
splice enhancer (ESE)
within the exon
RNA processing – mRNA
• mRNA processing –
Splicing
• 2. U2 recruits U4/U5/U6
trimer
• U6 replaces U1, U1 and U4
released
• U5 binds to upstream exon
RNA processing – mRNA
• mRNA processing –
Splicing
• 3. U6 catalyzes two
important reactions
– Cleavage of upstream exon
from intron (bound to U5)
– Lariat formation with A bulge
on intron
• Exons are ligated
• U2/U5/U6 remain with intron
RNA processing – mRNA
• mRNA processing – Splicing
• Several lines of evidence suggest that it is the RNA in
the snRNP that actually catalyzes the splicing reactions
– 1. Pre-mRNAs are spliced by the same pair of chemical
reactions that occur as group II (self-splicing) introns
– 2. The snRNAs needed for splicing pre-mRNAs closely
resemble parts of the group II introns
• Proteins likely serve supplemental functions
–
–
–
–
1. Maintaining the proper 3D structure of the snRNA
2. Driving changes in snRNA conformation
3. Transporting spliced mRNAs to the nuclear envelope
4. Selecting the splice sites to be used during the processing of
a particular pre-mRNA
RNA processing – mRNA
• mRNA processing –
Splicing
• Group II intron self-splicing
summary (rare)
RNA processing – mRNA
• Implications of RNA catalysis and splicing
• The RNA world
– Which came first, DNA or protein?... Apparently, it could have been
RNA
– Information coding AND catalyzing ability
• Alternative splicing
– Allows one gene to encode multiple protein products
• Intron sequences actually encode some snoRNAs
• Evolutionary innovation
– Exon shuffling
RNA processing - rRNA
• Eukaryotic ribosomes have four
distinct rRNAs:
– Three rRNAs in the large subunit
(28S, 5.8S, 5S in humans);
– One in the small (18S in humans)
subunit
– S value (or Svedberg unit)
•
•
•
•
28S = ~5000 nucleotides
18S = ~2000 nucleotides
5.8S = ~160 nucleotides
5S = ~120 nucleotides
– RNA pol I transcribes all but 5S
– 5S is transcribed by RNA pol III
Ribosomal RNA - rRNA
• Ribosomes are the location of
protein synthesis
– They are combinations of protein
and RNA and are made up of two
parts (small and large subunits)
• Millions exist in any given
eukaryotic cell
• ~80% of RNA in a cell is rRNA
• rDNA, typically exists in
hundreds of tandemly repeated
copies
RNA processing - rRNA
RNA processing - rRNA
• The likely rRNA processing pathway
– Cleavages 1 and 5 remove the ends of the 1° transcript
– Two pathways exist for the remaining processing
– End result is the same –
• 18S + paired 28S/5.8S
– 5S is produced by a second transcription unit
Review from last time
• The splicosome is a complex of RNA and protein units
responsible for splicing of immature mRNAs
• Be able to describe the functions of each snRNP
• The RNA portion of the snRNPs binds to the mRNA and
to other snRNPs and actually catalyzes the splicing
• The protein in the snRNPs serves other structural and
functional roles
• Ribosomal RNA is transcribed as a long unit but later
chopped up to its constituent parts
• The constituents along with proteins make up the small
and large ribosomal subunits
RNA processing – 5S RNA
• 5S rRNA
• Transcribed by RNA pol III
• Pol III is unique in that it utilizes promoters within the transcription
unit
RNA processing - tRNA
• Transfer RNAs (tRNA)
• Responsible for carrying amino acids to the site of
protein synthesis
• In humans, ~1300 genes for ~50 tRNAs
• Human tRNA genes exist on all chromosomes except 22
and Y and are highly clustered on 1, 6, and 7
• Transcribed by RNA pol III
RNA processing
• Small noncoding RNAs and RNA silencing
• To study the effect of disabling a gene,
researchers have had to produce ‘knockouts’
through a difficult, time consuming process
involving some random chance.
• …until the discovery of RNA interference
– introduce dsRNA for the gene to be silenced and the
mRNAs for that gene are destroyed
10_38_ES.cells.jpg
…until the discovery of RNA interference
introduce dsRNA for the gene to be silenced and the
mRNAs for that gene are destroyed
RNA processing
• Mechanisms of RNA
interference (siRNAs)
• Dicer – RNA endonuclease
• One of the RNA strands is
destroyed, the other acts to
identify the target mRNA
as part of RISC complex
• Slicer – RNA
endonuclease portion of
RISC
• Likely a defense against
foreign DNA
RNA processing
• MicroRNAs (miRNA)
• Work via a similar
mechanism
• Different source
• Synthesized by RNA pol II
• Later cleaved by dicer
• Block translation
Translation
• By the end of this series of slides, you should be able to
explain much of this animation
• http://www.as.wvu.edu/~dray/219files/Translation_588x392.swf
• An alternate animation is also provided:
http://www.as.wvu.edu/~dray/219files/TranslationAdvanced.wmv
Translation
• The genetic code
• Amino acids in a protein are
determined by a degenerate,
triplet code
• The code was determined
using synthetic RNAs
• The first, poly(U) ->
polyphenylalanine
• The genetic code is nearly
universal
Translation
•
•
•
•
The genetic code
Codon assignments are nonrandom;
Codons for same amino acid tend to be similar
Benefits:
– Less likely for a mutation to alter the amino acid sequence
• Synonymous vs nonsynonymous mutations
– Amino acids with similar chemical properties are encoded by
similar codons
Translation
• Translation - converting the nucleic acid information to
amino acid information
• A. Each tRNA is linked to a specific amino acid
• B. Each tRNA is also able to recognize a particular
codon in the mRNA
• C. Interaction between successive codons in mRNA &
specific aa-tRNAs leads to synthesis of polypeptide with
an ordered amino acid sequence
Translation
• tRNA characteristics
• 1. All are relatively small with similar numbers of
nucleotides (73 – 93)
• 2. All have a significant number of unusual bases made
by altering normal base posttranscriptionally
• 3. All have base sequences in one part of molecule that
are complementary to those in other parts
• 4. Thus, all fold in a similar way to form cloverleaf-like
structure (in 2 dimensions)
• 5. Amino acid carried by the tRNA is always attached to
A (adenosine) at 3' end of molecule
• 6. Unusual bases concentrated in loops where they
disrupt H bond formation; also serve as potential
recognition sites for various proteins
Translation
• Codon – Anticodon pairing
• Similar to typical basepairing but allows for third position
wobble
• The first two positions must pair exactly but the third is
more relaxed
• Anticodon U can pair with A or G on mRNA
• Anticodon I (derived from G) can pair with U, C, or A
• Allows for fewer required tRNAs
– Leucine (6 codons) requires only 3 different tRNAs
Translation
• tRNA activation
• Aminoacyl-tRNA synthetase (aaRS) guides
charging of tRNAs with amino acids
• ~20 different versions for the 20 different aa’s
Translation
• Ribosome structure
• Each ribosome has 3 sites
for association with tRNAs;
the sites receive each tRNA
in successive steps of
elongation cycle
– A (aminoacyl) site – P (peptidyl) site
– E (exit) site -
• A channel for the nascent
polypeptide to exit is also
present
Translation
• Ribosome structure
• tRNAs bind within these sites & span the gap between
the 2 ribosomal subunits
– The anticodon ends of the bound tRNAs contact the small
subunit, which plays a key role in decoding the information
contained in the mRNA
– The amino acid-carrying ends of bound tRNAs contact the large
subunit, which plays a key role in catalyzing peptide bond
formation
Review from last time
• 5S rRNA and tRNAs are transcribed by RNA pol III
• RNA pol III is unique in its use of an internal promoter
• siRNAs and miRNAs are RNAs involved in shutting
down a gene’s function without affecting the gene itself
• The genetic code is degenerate
• tRNAs are short RNAs that bridge the gap between
information in the mRNA and amino acid chain
• The structure of a ribosome is such that three sites are
formed, A, P, and E
• As mRNA threads through the ribosome the information
encoded is translated to form an amino acid chain
Translation
• Initiation of translation
•
•
•
•
Step 1. Bind the initiation codon
(AUG, met) to the small ribosomal
subunit
In bacteria
The Shine-Dalgarno sequence on
mRNA is complementary to 16S rRNA
Initiation Factors
–
–
–
IF1 – attaches 30S subunit to mRNA
IF2 – required for attachment of first tRNA
IF3 – likely prevents bind of large subunit
Translation
• Initiation of translation
•
•
Step 2. Bind the first tRNA (tRNAMet)
Enters the P site with the help of IF 2
Translation
• Initiation of translation
•
•
Step 3. Bind the large subunit
IFs released and large subunit binds
Translation
• Initiation of translation
•
•
•
•
•
•
Bind the initiation codon (AUG, met) to the small ribosomal subunit
In eukaryotes
Three IFs + tRNAMet bind to 40S subunit
Separately mRNA binds to additiona IFs and PABP
These components come together and scan along mRNA until AUG is
reached
Large subunit binds
Translation
• Elongation
• The players – EF-Tu/GTP/tRNA
complex
– EF-Tu – escorts the tRNA to the
A site
– GTP – provides energy
– The tRNA - duh
• Any tRNA can enter but only
the correct one will induce the
conformational changes to
induce binding to mRNA
• Once in, GTP -> GDP and TuGDP is released
Translation
• Elongation
• Peptide bond is formed
between aa’s
• Peptidyl transferase – a
ribozyme
• tRNA in P site is now
uncharged
Translation
• Elongation
• Translocation of the ribosome
along the mRNA (3 nt)
• tRNAs rachet positions
• EF-G induced
• GTP -> GDP + P required
Translation
• Elongation
• Release of the uncharged
tRNA and repeat the whole
cycle
• ~1/20 second
Translation
• Termination
• Three codons (UAA, UGA, UAG) have no
complementary tRNAs
• Protein released when one is reached
• Release factors are required
• Bacteria RF1, RF2, RF3
• Eukaryotes eRF1, eRF3
• Each recognizes particular stop codon much like a tRNA
• RF3/eRF3 binds GTP to energize the release of the
polypeptide and disassemble the ribosome
• The complete process (for bacteria) is illustrated using
videos on the class website.
Translation
• Polyribosomes
Prokaryote
Eukaryote
Note the difference –
Due to presence/absence of
nuclear membrane
• http://www.as.wvu.edu/~dray/219files/Protein_Synthesis
%20_Translation_2008.avi