Transcription unit may produce several mRNA (RNA processing); one mRNA → translated only one protein

(1)

Chapter 4 Part II

In prokaryotic cell

One operon = one transcript unit Transcript unit: specific one promoter and

termination site, and contain several gene

one mRNA → may translated several protein

In eukaryotic cell

Most gene expressed from separate transcription unit (discontineous)

Transcription unit may produce several mRNA (RNA processing); one mRNA → translated only one protein

Transcription unit has two

types: simple and complex

(2)

Simple and complex eukaryotic transcription

Mutation control region: no mRNA expression → no protein → no function

Mutation Exon : mRNA expression (some wrong) → abnormal protein → activity change Finally, translated one protein

For gene that are transcribed from different promoters (regulator factor) in different cell type

Multiple mRNA transcribe from primary transcript

Finally, translated more one protein

Protein-coding genes may be solitary or belong to a gene family

Solitary gene: in multicellular organism, 20-50% protein coding gene are represented

only once in the haploid (a simple transcription unit) Duplicated gene: gene family → protein family homologous

duplicated gene encode protein with similar

(3)

RNAi - RNA interference

siRNA- active molecules in RNA interference; degrades mRNA (act where they originate)

miRNAs - tiny 21–24-nucleotide RNAs; probably acting as translational regulators of protein-coding mRNAs

stRNA - Small temporal RNA; (ex. lin-4 and let-7 in

Caenorhabditis elegans

snRNA - Small nuclear RNA; includes spliceosomal RNAs (processing)

snoRNA - Small nucleolar RNA; most known snoRNAs are involved in rRNA modification

New Roles of RNA

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

can be produced by same transcript

Production of heavy chain genes in mouse by recombination of V, D, J, and

C gene segments during development

(4)

Differences Between Transcription In Prokaryotes and Eukaryotes

Transcription And Translation In Prokaryote---the same time Eukaryotic Transcription and translation---different time

Processing Eukaryotic mRNA

Different cell → different transcription

In prokaryotic cell

One operon = one transcript unit Transcript unit: specific one promoter and

termination site, and contain several gene

one mRNA → may translated several protein

(5)

In eukaryotic cell

Most gene expressed from separate transcription unit (discontineous)

Transcription unit may produce several mRNA (RNA processing); one mRNA → translated only one protein

Transcription unit has two types: simple and complex

Simple and complex eukaryotic transcription

Mutation control region: no mRNA expression → no protein → no function

Mutation Exon : mRNA expression (some wrong) → abnormal protein → activity change Finally, translated one protein

For gene that are transcribed from different promoters (regulator factor) in different cell type

Multiple mRNA transcribe from primary transcript

Finally, translated more one protein

different cell different transcription.swf

(6)

接第七章 7.1 and 7.2

Transcriptional control of gene expression

Key players 1. Promoter 2. RNA polymerase

3. Transcription factor (regulator) 4. Operator (or enhancer)

In prokaryotic cell

In eukaryotic cell: DNA high condense → need decondense→ open

轉到ch6 p216

The structure of genes and chromosomes

Transcription initiation

Overview of eukaryotic transcription control

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

(7)

Transcription initiation by bacterial RNA polymerase requires association with a sigma factor

Operons: each of encodes enzymes gene involved in a particular metabolic pathway or protein

All the gene are coordinately regulated: that is they are all activated or repressed (要就全部表現,不然全部不表現)

Initiate stage: RNA polymerase bind sigma factor (σ70) → σ 70 recognize to promoter sequence (6 base pair) about -10 → - 10 to - 35 is promoter region →RNA polymerase plus σ70 bind it (RNA p is bind to -50 to 20)→ interact with DNA double strand →

σ 70 is initiation factor

→→→→→

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

σ⁷⁰subunit 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)

Lac repressor-operator interactions

operator

Strong promoter: a high rate Weak promoter: a low rate

(8)

請結合p279

RNA polymerase (RNAP) vs. Transcription

Three eukaryotic polymerases catalyze formation of different RNAs

•α-amanitin from Amanita Phalloides binds tightly to RNA Pol II and blocks transcriptional elongation.

• RNA Pol I transcribe 1 gene at ~200 copies. The gene for the 45S pre-rRNA is present in tandem array.

• RNA Pol II transcribe ~25,000 genes;

• RNA Pol III transcribe 30-50 genes at variable copy numbers.

DNA affinity chromatography for purification

(9)

Similar, but Yeast RNAPII more complex than bacterial

CTD;

carboxyl- terminal domain

Comparison of 3-D structures of bacterial and eukaryotic RNA polymerases

Try-Ser-Pro-Thr-Ser-Pro-Ser:

CTD, repeat sequence.

Phosphorylation → RNAP II → initiate transcript

Schematic representation of the subunit structure of E. Coil RNA core polymerase and yeast nuclear RNA polymerase

All three yeast polymerases have five core subunits that exhibit some homology with the β, β‘, α and ω subunits in E. coli RNA polymerase.

RNA polymerases I and III contain the same two non-identical α-like subunits, whereas polymerase II has two copies of a different α-like subunit.

All three polymerases share four other common subunits. In addition, each RNA polymerase contains three to seven unique smaller subunits.

The largest subunit (1) of RNA polymerase II also contains an essential C-terminal domain (CTD). 27 (yeast) to 52 (human) copies of (YSPTSPS).

Phosphorylation of CTD is important for transcription and RNA processing.

Antibody staining demonstrates that the CTD of RNAP II is phosphorylated during in vivo transcription

Green: unphosphorylated Red: phosphorylated

Puffed region The region of

geomes is very actively transcribed .

αΙ

β β’

αΙΙ

σ

⁷⁰

RNAP HOLOENZYME -σ

⁷⁰

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

(10)

α α

₂

α

₂

β α

₂

ββ’ = core enzyme αΙ

β β’

αΙΙ

CORE ENZYME

Sequence-independent, nonspecific transcription initiation

+

vegetative (principal σ)

σ

⁷⁰ SIGMA SUBUNIT

interchangeable, promoter recognition

The assembly pathway of the core enzyme

heat shock (for emergencies)

σ

³²

nitrogen starvation (for emergencies)

σ

⁶⁰

RNA polymerase II initiates transcription at DNA sequence corresponding to the 5’ cap of mRNA

Need 5’ cap for efficient translation

The mRNA transcription-initiation machinery

Proteins associate into multimeric structures and macromolecular assemblies

ch3

(11)

p.297

TBP: TATA box-binding protein TF: transcription factor

TBP (TATA-box binding protein)

•Conserved C-terminal domain of 180 amino acids.

•A monomer with a saddle-shaped structure;

the two halves show an overall dyad symmetry but are not identical.

•Binds multiple transcription factors (TAFs, TFIIB and TFIIA).

•Binds in the minor groove and significantly bends DNA.

(12)

Model for the structure of and RNAP II preinitiation complex

色素性乾皮病 xeroderma pigmentosum

skin and the tissue covering the eye is extremely sensitive to the ultraviolet part of sunlight; similar to cancer

TFIIH mutation

柯凱因氏症候群 Cockayne syndrome

嚴重的外貌與智商退化

In vivo transcription initiation by RNAP II requires additional proteins

0402_transcript_mechanism.swf

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 (方向)

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

(13)

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)

Æ lac operon is also regulated by glucose levels [glucose] = high Æ Low transcription of lac operon [glucose] = low Æ 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 Æ [cAMP] = high [glucose] = high Æ [cAMP] = low

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 Æ low level of transcription occurs

• Glucose present Æ cAMP is low Æ CAP does not ‘help’ transcription and thus it remains at low level

3) Lactose present; no glucose

• The presence of lactose inactivates the repressor Æ Transcription occurs

• NO Glucose Æ cAMP is high Æ cAMP binds CAP (becomes activated) Æ CRP binds & ‘Helps’ Transcription

• High Level of transcription

Levels of Control of Lac Operon Expression 3 Scenarios 情節:

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

For example:

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 σ

⁵⁴

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 one of RNA polymerase subunit, is regulated solely (獨一) 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

Phosphorylation: 在蛋白質ser, tyr, thr的位置接上磷酸根

(14)

σ54 binds to the glnA promoter (did not melt DNA and transcription) → bind with NtrC and enhancer → turn on glnA → glutamine synthetase mRNA → transcription ↑

glnA →glutamine synthetase → synthesis gltamine

Normal condition: glutamine ok

Low glutamine:

NtrB phosphorylates NtrC → NtrC conformational change →binds to enhancer upstream of the glnA promoter.

NtrC and NtrB are regulatory protein for transcription.

NtrB

蛋白質或酶被磷酸化過程會改變其構形導活性改變

Nitrogen Regulatory Protein C (NtrC) plays a central role in the bacterial metabolism of nitrogen

N-terminal receiver

domain

Central catalytic domain

DNA binding domain

Experimental evidence for

population shift

(15)

Asp54 Phosphate

Changing nitrogen levels promote the activity of NtrB kinase

NtrB kinase phosphorylates NtrC at aspartate 54 in the receiver domain

Protein conformational change

Asp54 Phosphate

Phosphorylation promotes conformational change in the receiver domain

Protein conformational change

• NtrC – active and inactive

conformations apparent

• P-NtrC – protein shifted towards activated

conformation

• Volkman, B.F. et al.

Science 291, 2429- 33 (2001)

Many bacterial responses are controlled by two-component regulatory system (NtrB, NtrC and PhoR, PhoB)

Low phosphate in the environment and periplasmic space

Conformational change When 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

(16)

The three roles of RNA in protein synthesis

Messenger RNA Transfer RNA Ribosomal RNA

再回到第四章

Roles of RNA

1. Information carrier: mRNA

2. Adaptor molecule:

tRNA

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

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 (協調) 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

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

(17)

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

普遍

Folded structure of tRNA promotes decoding (解譯) functions

DNA (4 nucleotide) → mRNA→ protein (20 types), need tRNA and aminoacyl-

tRNA synthetase (very specific)

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); many amino acid have more than one tRNA;

many tRNA pair with more codons

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 (苜蓿) 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 Wobble Hypothesis (position)

(18)

3D structures of RNA : transfer-RNA structures

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

CU G

U 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

acceptor stem tRNA

anticodon

acceptor stem

tRNA

^Phe

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.

(19)

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 tRNA. 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

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.

inosine

I = inosine which is sometimes found in tRNA

adenine deaminated → inosine (I) → form nonstandard base pairs.

Wobble base pairs

Inosine Uracil

(20)

The Wobble Hypothesis

The first two bases of the codon make normal (canonical 根據教法) 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

OH OH

H H

H CH2

H O P O P O

−O P O

O⁻ O⁻

O O

O⁻ R

H C C NH₃⁺

O⁻ O

O

OH OH

H H

H CH2

H O P O C

O

O⁻ H

C R

NH₂ O

Adenine

Adenine ATP

Amino acid

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

OH OH

H H

H CH2

H O P O C

O

O⁻ H

C R

NH2

O

Adenine

O

OH O

H H

H CH2

H O P O

O

O⁻

Adenine tRNA

C HC

O

NH₃⁺ R

tRNA AMP Aminoacyl-AMP

Aminoacyl-tRNA (terminal 3’nucleotide of appropriate tRNA)

3’ 2’

2

Aminoacyl-tRNA Synthetase - summary:

1. amino acid + ATP Æ aminoacyl-AMP + PP

_i

2. aminoacyl-AMP + tRNA Æ aminoacyl-tRNA + AMP

The 2-step reaction is spontaneous overall, because the concentration of PP

_i

is kept low by its hydrolysis, catalyzed by Pyrophosphatase.

Activate amino acids by covalently linking them to

tRNAs

(21)

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

E. coli 70S 30S

16S RNA 21 proteins

50S 23S & 5S

RNAs 31 proteins Rat

cytoplasm

80S 40S

18S RNA 33 proteins

60S 28S, 5.8S, &5S

RNAs 49 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