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PromotersPromoters -- DNA sequences that guide DNA sequences that guide RNA polymeraseRNA polymerase to the beginning of to the beginning of a gene (transcription initiation site).a gene (transcription initiation site).
TerminatorsTerminators -- DNA sequences that specify then termination of RNA synthesis DNA sequences that specify then termination of RNA synthesis and release of RNAP from the DNA.and release of RNAP from the DNA.
RNA Polymerase (RNAP)RNA Polymerase (RNAP) -- Enzyme for synthesis of RNA.Enzyme for synthesis of RNA.
Reaction (ordered series of steps)Reaction (ordered series of steps) 1) Initiation.1) Initiation. 2) Elongation. 2) Elongation. 3) Termination.3) Termination.
Bacterial (Prokaryotic) Transcription
rNTP vs.dNTP

About 8 base pair For continuous RNA synthesis and without dissociation
Two mechanisms 1) Rho - the termination factor protein
– rho is an ATP-dependent helicase – it moves along the RNA transcript, finds the "bubble",
unwinds it and releases the RNA chain.
Termination of transcription
2) Rho-Independent - termination sites in DNA
inverted repeat, rich in G:C, which forms a stem-loop in RNA transcript
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Rho-Dependent Transcription Termination (depends on a protein AND a DNA sequence)
G/C -rich site
RNAP slows down
Rho helicase catches up
Elongating complex is disrupted
Two mechanisms 2) Rho-Independent
- termination sites in DNA – inverted repeat, rich in G:C, which forms a
stem-loop in RNA transcript
Rho-Independent Transcription Termination (depends on DNA sequence - NOT a protein factor)
Stem-loop structure
• The pause may give the hairpin structure time to fold
• The fold disrupts important interactions between the RNAP and its RNA product
• The U-rich RNA can dissociate from the template
• The complex is now disrupted and elongation is terminated
DNA RNA A T T U G C C G
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Current model of bacterial RNA polymerase bond to a promoter
templateStructure of RNA polymerase
RNA polymerase are similar in eukaryotic and prokaryotic cell
Five subunit: 2 large subunit: β, β’; 2 smaller subunits α and ω (only Stabilizes and assembly of its subunits)
Mg2+
Only a single RNA polymerase (prokaryotic)
In E.coli, RNA polymerase is 465 kD complex, with 2 α, 1 β, 1 β', 1 σ
β' binds DNA β binds rNTPs and interacts with σ β and β ' together make up the
active site α subunits appear to be essential for
assembly and for activation of enzyme by regulatory proteins
RNA Polymerase • RNA Polymerase is a spectacular () enzyme, functioning in: 1 Recognition of the promoter region 2 Melting of DNA (Helicase + Topisomerase); unwinding DNA 3 RNA Priming (Primase) 4 RNA Polymerization; add rNTP 5 Recognition of terminator sequence
RNA-DNA hybrid Length? 3 to 9 bases, it is short and transit
In Bacterial which can hold~16 bp In yeast which can hold ~25 bp
Thus, RNAP is a multisubunit enzyme
In Bacteria (simple system) - all three classes are transcribed by the same RNA polymerase (RNAP for short)
In Eukaryotes (complex system) - each class is transcribed by a different RNA Polymerase ••RNAP I RNAP I -- rRNAsrRNAs ••RNAP II RNAP II -- mRNAsmRNAs ••RNAP III RNAP III -- tRNAstRNAs & small ribosomal & small ribosomal RNAsRNAs
••Remember: only RNAP did not transcript !!!! Need many Remember: only RNAP did not transcript !!!! Need many transcription factor (protein)transcription factor (protein)
FlashFlash--22
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One model for transcriptional activation Gene Regulation Protein complex → DNA → open/tight DNA → transcription
Transcription is regulated by proteins binding to or near promoters – Three types of proteins involved:
• Specificity factors • Repressors • Activators
– Repressors: bind to specific sites on DNA • Called operators • Either near or overlapping the promoter • Block movement of RNA-polymerase
-Activators: bind to specific sites on DNA, help RNAP moving
Operon: arrangement of genes in a functional group
Organization of genes differs in prokaryotic and eukaryotic DNA
In prokaryotic: 1. logic: genes devoted () to a
single metabolic goal; protein synthesis from a contiguous array in DNA. It means that one gene → one protein.
2. Arrangement of genes in a functional group is cell an operon, because it operate as a unit from a single promoter. One promoter → one gene → one protein
3. The genes are closely packed with very few non-coding gaps
DNA → direct to co-linear mRNA → → translated protein
Genomes
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corresponding DNA sequence 2. DNA contain exons (coding
sequence) and introns (non-protein- coding segments)
3. DNA → RNA, remove introns and carefully stitched back together to produced many mRNAs
4. Functional (mature) mRNA from precursor mRNA processed (splicing)
5. DNA → pre-mRNA → splicing → mature mRNA→ protein → add modification → mature protein
Eukaryotic precursor mRNA are processed to form functional mRNAs
Tryptophan metabolite enzyme Tryptophan (trp)
Each gene is transcripbed from its own promoter
In prokaryotic, RNA synthesis can occur in 5’ or 3’ end of DNA; transcription and translation can occur at the same time.
In eukaryotic, in nucleus DNA → transcription → precursor mRNA → procession → functional mRNA → transport to cytoplasm → translated to protein; Transcription and translation are in different time and place.
Pre-mRNA are modified at the tow ends, and keep in mRNA. It can protect the degradation of RNA form nucleus to cytoplasm. Don’t need DNA template.
Modification of 5’ end: by RNA polymerase II → add 5’cap; methylation
Modification of 3’ end: by poly A polymerase, add 100-250 A and produced poly A tail.
mRNA processing – RNA splicing, 5’ and 3’ retain noncoding regions (untranslated regions; UTRs).
In mammalian mRNA, 5’ UTR about >100 nucleotides, 3’ UTR about several kilobases
The ribose of the second nucleotide also is methylated
Alternative RNA splicing increase the number of proteins expressed from a single eukaryotic gene
One gene → RNA splicing → different RNA→ different protein
Isoform: by alternative splicing production of different forms of a protein.
Untranslated region One gene can lead to more than one
protein (e.q. antibodies)
Formed three protein-coding exon
Exons: part of the gene that is expressed. Introns: part of gene that is spliced out
from pre-mRNA.
RNA Processing:
concurrent.
(Protein synthesis) are separated.
• 5’ cap is added to 5’ nucleotide; m7Gppp (Stability)
• String of adenylic acids are added to the 3’ end (Poly
A tail)
by ligation of coding exons
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Add a GMP. Methylate it and 1st few nucleotides
Cut the pre-mRNA and add A’s
Functions of 5’ cap and 3’ polyA Both cap and polyA contribute to stability of mRNA:
– Most mRNAs without a cap or polyA are degraded rapidly.
– Shortening of the polyA tail and decapping are part of one pathway for RNA degradation in yeast.
Need 5’ cap for efficient translation:
– Eukaryotic translation initiation factor 4 (eIF4) recognizes and binds to the cap as part of initiation.
– Assists mRNA export to the cytoplasm
Cell type specific splicing of fibronectin pre-mRNA
Alternative RNA splicing increases the number or proteins expressed from a single eukaryotic gene
Higher eukaryote have multidomain tertiary structure only from a small number of exons.
Single gene →Multiple introns→alternative splicing → protein isoforms
Alternative splicing: The presence of multiple introns in many eukaryotic genes permits expression of multiple, related proteins form a single gene.
> 20 isoforms fibronectin from different alternatively spliced mRNA
• Alternative splicing – Different mRNAs
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Production of heavy chain genes in mouse by recombination of V, D, J, and
C gene segments during development
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Eukaryotic Transcription and translation--------------different time
Processing Eukaryotic mRNA
Functions of 5’ cap and 3’ polyA Both cap and polyA contribute to stability of mRNA:
– Most mRNAs without a cap or polyA are degraded rapidly.
– Shortening of the polyA tail and decapping are part of one pathway for RNA degradation in yeast.
Need 5’ cap for efficient translation:
– Eukaryotic translation initiation factor 4 (eIF4) recognizes and binds to the cap as part of initiation.
RNA Processing:
concurrent.
(Protein synthesis) are separated.
• 5’ cap is added to 5’ nucleotide; m7Gppp (Stability)
• String of adenylic acids are added to the 3’ end (Poly
A tail)
by ligation of coding exons
Different cell → different transcription
Control of gene expression in prokaryotes
Repressed: the corresponding mRNA and encoded protein are synthesis at low rates Activated: at high rates Operator: in DNA, might have activated and repressed. Determined by activator
and repressor (a DNA binding protein) Highly regulated in order to adjust the cell’s enzymatic machinery and structural
components to changes in the nutritional and physical environment. The lac operon in E. coli as primary example. Operon is transcribed from one start site into a single mRNA, all the genes within
an operon are coordinately regulated.
Repressors - which inhibit transcription by binding to an ‘operator’ DNA sequence near the promoter
- which can be modulated by Effectors Co-repressors
Activators - which enhance transcription by binding a DNA sequence near the promoter sequence
- these may or may not be modulated by an effector
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Schematics of (a) positive regulation of gene transcription and (b) negative regulation of gene transcription
Initiation of lac operon transcription can be repressed and activated σ70 subunit of RNA polymerase
bind to the lac promoter, it upstream of the start site.
When no lactose (or low concentration), lac repressor bind to operator, which overlaps the transcription start site → block polymerase bind
When lactose increase → lactose bind to lac repressor → lac repressor conformation change →lac repressor can not bind operator →transcription start exposure → polymerase bind → transcription easy
Glucose overuse → cAMP↑→ bind to CAP protein → conformation change →interaction with polymerase → transcription ↑
Catabolite Activator Protein (CAP)
αΙ
+ vegetative
The assembly pathway of the core enzyme
heat shock (for emergencies)
RNAP HOLOENZYME -σ70
Promoter-specific transcription initiation
In the Holoenzyme:
· β' binds DNA · β binds NTPs · β and β ' together make up the active site · α subunits appear to be essential for assembly and for activation of enzyme by regulatory proteins. They also bind DNA. · σ recognizes promoter sequences on DNA
Positive and Negative Regulation in the lac Operon
Operon has three structural genes: lacZ, lacY, lacA lacI gene is upstream and in the opposite orientation ()
Catabolite Activator Protein (CAP) is a positive regulatory protein and cyclic AMP is its inducer molecule.
Lac Repressor (lacI gene) is a negative regulatory protein, and lactose (or IPTG) is its inducer molecule.
Lac repressor as Tetramer binds two regions of the DNA Forms a loop of DNA betewwn the two binding sites Binding prevents transcription Allosteric enzyme with binding site for allolactose affecting one dimer of
the tetramer
The lac operon is also subject to POSITIVE regulation, by CAP in the presence of cAMP
The lac operon is induced by the presence of lactose in the medium, but E. coli prefers to use glucose (better energy source)
lac operon is also regulated by glucose levels [glucose] = high Low transcription of lac operon [glucose] = low High transcription of lac operon
This correlates to the activity of CAP which activates lac operon in the presence of cAMP (cyclic AMP)
[glucose] = low [cAMP] = high [glucose] = high [cAMP] = low
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1) No Lactose around • Operon switched off, essentially no mRNA regardless of [glucose]
2) Lactose present; glucose also present • The presence of lactose inactivates the repressor
low level of transcription occurs • Glucose present cAMP is low CAP does not ‘help’ transcription
and thus it remains at low level 3) Lactose present; no glucose
• The presence of lactose inactivates the repressor Transcription occurs
• NO Glucose cAMP is high cAMP binds CAP (becomes activated) CRP binds & ‘Helps’ Transcription
• High Level of transcription
3 Scenarios :
Lac operon flash
Small molecules regulate expression of many prokaryotic gene via DNA- binding repressor
Specific repressor binds to operator → blocking transcription initiation
Small molecules, called inducer, binds to repressor → controlling DNA-binding
activity and transcription rats
Tryptophan: when trp high → bind to trp repressor → conformational change →
repressor easy bind to operator → transcription low
lac operon: inducer lactose; lactose bind to lac repressor → conformational
change → bind to operator hard → transcription high
Transcription by σ54 RNA polymerase is controlled by activator that bind far from the promoter σ70 is major form of the bacterial enzyme. Transcription of certain groups of genes, is carried out by RNA polymerases
containing one of several alternative sigma factors the recognized different consensus promoter sequences than σ70.
σ54 is also RNA polymerase, is regulated solely () by activators whose binding sites in DNA. Referred to as enhancer, it can activate transcription.
For example: NtrC (nitrogen regulatory protein C)-stimulates transcription from the
promoter of the glnA gene (encodes glutamine synthetase )
Autokinase Sensor Proteins (NtrB) (autophosphorylation) – Sensor domain – Transmitter domain (C-terminus)
Response Regulators (NtrC) – N-terminal receiver domain – Cross-regulation
σ54 binds to the glnA promoter (did not melt DNA and transcription) → bind with NtrC and enhancer → turn on glnA → glutamine synthetase mRNA → transcription ↑
Low glutamine: NtrB phosphorylates NtrC → binds to
enhancer upstream of the glnA promoter.
NtrC and NtrB are regulatory protein for transcription.
glnA →glutamine synthetase → synthesis gltamine
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Low phosphate in the environment and periplasmic space
Conformational changeWhen external environment phosphate↓→ periplasmic space phosphate ↓→ PhoR can not bind phosphate → Conformational change and dissociate → kinase domain exposure → transfer ATP phosphate → to Pho B → transcription The second protein also called a response regulator. One PhoR can phosphorylated many PhoB
Like NtrB
Like NtrC
Messenger RNA Transfer RNA Ribosomal RNA
Roles of RNA
3. Catalyst and structural molecule: rRNA
4. Viral genomes: Some viruses use RNA as their genetic material
Transfer RNA (tRNA) All types of RNA, including tRNA, are transcribed from template DNA In Eukaryotes, each tRNA can be used repeatedly
tRNA is a single-stranded RNA only about 80 nucleotides. 45 distinct types of tRNA, some tRNAs recognize two or more mRNA codons
specifying the same AAs.
The enzymes that catalyzes the attachment of an AA to its tRNA Each of the 20 AAs has a specific aminoacyl-tRNA synthetase Attachment of the AA to tRNA The aminoacyl-tRNA complex release from the enzyme and transfers its AA to a growing polypeptide chain
Ribosomal RNA (rRNA)
Ribosomes coordinate () the pairing of tRNA anti-codons to mRNA codons
Two subunits (small and large) 60% rRNA and 40% protein Both subunits are constructed in the nucleolus: once in the cytoplasm, are
assembled into functional ribosomes when attached to an mRNA
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Messenger RNA (mRNA)
Carries the genetic information transcribed from DNA in the form of a series of three nucleotide sequences, called codons, each of which specifies a particular amino acid.
Genetic code Of the 64 possible codons in the genetic code, 61 specify individual amino acids and three are stop codons.
Reading frame: the sequence of codons hat runs from a specific start codon to a stop codon
Start codon: most is AUG start (initiator) codon-methionine in eukaryote. Stop codon: UAA, UGA, UAG Reading frame: the sequence of codons that runs from a specific start codon to a stop codon. In eukaryote, exon = reading frame
Each codon is the same in most known organisms But some exceptions to the general code probably were later evolutionary
developments
Folded structure of tRNA promotes decoding () functions
DNA (4 nucleotide) → mRNA→ protein (20 types), need tRNA and aminoacyl- tRNA synthetase
enzymes that catalyzes the attachment of an AA to its tRNA Each of the 20 AAs has a specific aminoacyl-tRNA synthetase
30-40 different tRNAs in bacterial, 50-100 in animal and plant, it more than the number of amino acids (20)
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The structure of tRNA
1. About 70-80 nucleotide long 2. The exact nucleotide sequence varies
among tRNA. All tRNA fold into a similar stem-loop arrangement in 2 dimensions (four base-pair stems and three loops), like cloverleaf ()
3. The four stems are short double helices stabilized by Watson-Crick base paring.
4. The loops about 7-8 nucleotides 5. The CCA sequence at the 3’ end is
found in all tRNAs 6. Attachment of amino acid to the 3’
end 7. Some A,U,C,G are modified and
located in specific region
Secondary structure of tRNA (cloverleaf)
Tertiary structure of tRNA
tRNA
Double helical stem domains arise from base pairing between complementary stretches of bases within the same strand. These stem structures are stabilized by stacking interactions as well as base pairing, as in DNA. Loop domains occur where lack of complementarity or the presence of modified bases prevents base pairing.
A U A C C U A U G G
C U
C U
stem loop
: : : : : RNA structure: Most RNA molecules have secondary structure, consisting of stem & loop domains.
The “cloverleaf” model of tRNA emphasizes the two major types of secondary structure, stems & loops. tRNAs typically include many modified bases, particularly in loop domains.
anticodon loop
There are 61 codons specifying 20 amino acids.
Minimally 31 tRNAs are required for translation, not counting the tRNA that codes for chain initiation.
Mammalian cells produce more than 150 tRNAs.
The wobble hypothesis (Crick, 1966)
In 1965, Holley determined the sequence of yeast tRNA(ala): he found the nucleotide Inosine at the 5’ end in the anticodon.
Nonstandard base pairing often occurs between codons and anticodons In perfect condition, codons and
anticodons cells would have to contain exactly 61 different tRNA species. Codons and anticodons might specific recognize
However, most cell contain fewer than 61. So broad recognition can occur.
Wobble position: broader recognition of nonstandard pairing between bases. The third of 3’ end base in mRNA and corresponding first base in its tRNA.
The first and second base of codon (mRNA) almost formed standard base pair.
adenine deaminated → inosine (I) → form nonstandard base pairs.
I similar to G, A
inosine
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The Wobble Hypothesis
The first two bases of the codon make normal (canonical ) H-bond pairs with the 2nd and 3rd bases of the anticodon
At the remaining position, less stringent rules apply and non-canonical pairing may occur
The rules: first base U can recognize A or G, first base G can recognize U or C, and first base I can recognize U, C or A (I comes from deamination of A)
Advantage of wobble: dissociation of tRNA from mRNA is faster and protein synthesis also
A tRNA can frequently recognize several codons because of non standard Watson-Crick hydrogen bonding between the nucleotides
This effect is called the wobble hypothesis
I = inosine which is sometimes found in tRNA
Aminoacyl-tRNA Synthetases catalyze linkage of the appropriate amino acid to each tRNA. The reaction occurs in 2 steps. In step 1, an O atom of the amino acid α-carboxyl attacks the P atom of the initial phosphate of ATP.
O
OHOH
HH
H
CH2
H
Aminoacyl-AMP
1
+ PPi
In step 2, the 2' or 3' OH of the terminal adenosine of tRNA attacks the amino acid carbonyl C atom.
O
OHOH
HH
H
CH2
H
OPOC
O
O−
3’ 2’
2. aminoacyl-AMP + tRNA aminoacyl-tRNA + AMP
The 2-step reaction is spontaneous overall, because the concentration of PPi is kept low by its hydrolysis, catalyzed by Pyrophosphatase.
Activate amino acids by covalently linking them to tRNAs
Ribosomes are protein-synthesizing machines Increase synthesis of protein about 2-5 aa /sec
3 subunits
4 subunits S: svedberg units, a measure of the sedimentation rate of suspendend particles centrifuged under standard conditions
Eukaryotic cytoplasmic ribosomes are larger and more complex than prokaryotic ribosomes. Mitochondrial and chloroplast ribosomes differ from both examples shown.
Ribosome Source
Whole Ribosome
Small Subunit
Large Subunit
21 proteins
Ribosome Composition (S = sedimentation coefficient)
Ribosomal RNA (rRNA) associates with a set of proteins to form ribosomes, structures that function as protein-synthesizing machines
Ribosomes Origin Complete
ribosome Ribosomal subunit
18 S 5 S
5.8 S 25 S
16 S 4.5 S 5 S 23 S
C. 24 C. 35
18 S 5 S
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Translation initiation usually occurs near the first AUG closest to the 5’ end of an mRNA
Initiation factor
eIF4A (helicase activity) →uses energy→ unwind RNA→complex move Kozak sequence: ACCAUGG
Protein synthesis: methionly-tRNAi recognizes the AUG start codon
Initiator factor: IF
• Initiation The inactive 40S and 60S subunits will bind to each other with high affinity to form
inactive complex This is achieved () by eIF3, which bind to the 40S subunit mRNA forms an
preinitiation complex (also bound eIF1A, Met-tRNAi Met, eIF, GTP) with a
ribosome; when eIF 2 phosphorylated → GDP/GTP exchange X→ translation X
A number of initiation factors participate in the process. Cap sequence present at the 5’ end of the mRNA is recognized by eIF4 complex,
eIF4 interaction with preinitation complex. Subsequently eIF3 is bound and cause the binding of small 40S subunit in the
complexes (initiation complex) step 2→ slide or scan; eIF4A plus mRNA → helicase→open RAN secondary structure (ATP dependent)
The 18S RNA present in the 40 S subunit is involved in binding the cap sequence eIF2 binds GTP and initiation tRNA, which recognize the start codon AUG → eIF2
GTP hydrolysis → irreversible step for further scanning. eIF4A unwind the RNA secondary structure by hydrolysis of ATP, 40S complex
migrate down stream until it finds AUG start codon eIF5 hydorlysis a GTP →The large 60S subunit is then bound to the 40S subunit It is accompanied by the dissociation of several initiation factor and GDP The formation of the initiation complex is now completed Ribosome complex is able to translate
Functions of 5’ cap and 3’ polyA
3D structures of RNA :transfer-RNA structures