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Mechanisms of Transcription

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The flow of genetic information

Gene: The region of DNA that

controls a discrete hereditary

characteristic of an organism,

usually corresponding to a single

protein or RNA.

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Genome size is related to the complexity of the organism

Species Genome size (Mb) Approximate

number of genes

Gene density

(genes/ Mb)

PROKARYOTES (bacteria)

Mycoplasma geni tali um 0.58 500 860

Streptococus pneumonia 2.2 2,300 1,600

Escheri chia coli 4.6 4,400 950

EUKARYOTES

Fungi: Saccharomyces cerevisiae 12 5,800 480Invertebrates: Caenorhabdi tis elegans 97 19,000 200

Invertebrates: Drosophil a melanogaster 180 13,700 80

Vertebrate: Homo sapiens 2,900 27,000 9.3

Vertebrate: Mus musculus 2,500 29,000 12

Plants: Arabidopsis thal iana 125 25,500 200

Plants: Oryza sativa (rice) 430 >45,000 >100

* Genome: the whole of the genetic information of an organism (one haploid set of

chromosomes in eukaryotes.

* Genome size: The length of DNA associated with one haploid complement of chromosome

* Gene: The region of DNA that controls a discrete hereditary characteristic of an organism,

usually corresponding to a single protein or RNA

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More complex organisms have decreased gene density

-Increases in gene size and increases in the DNA between genes, called

intergenic sequences.- Individual genes are longer because of two reasons: 1) increase in regionss of

DNA required to direct and regulate transcription, called regulatory sequenes;

2) protein-encoding genes in eukaryotes frequently have discontinuous protein-

coding regions. These interspersed non-protein-encoding regions is called

introns.

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Genes can be expressed with different efficiency

- Many identical RNA copies can made from the same gene, and each RNA

molecule can direct the synthesis of many identical protein molecules.

- Each gene can be transcribed and translated with a different efficiency. Gene A is

transcribed and translated much more efficiently than is gene B. This allows the

amount of protein A in the cell to be much higher than that of protein B.

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Portions of DNA Sequence Are Transcribed into RNA

Transcription:Copying of one strand of DNA into a complementary

RNA sequence by the enzyme RNA polymerase

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The chemical structure of RNA differs slightly from that of DNA

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Transcription produces RNA complementary to one strand of DNA

Uracil forms base pair with Adenine

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The enzymes that carry out transcription are called

RNA polymerase

- RNA polymerases catalyze the formation of the phosphodiester bonds that link thenucleotides together and form the sugar-phosphate backbone of the RNA chain.

- RNA synthesis (Transcription) requires ribonucleoside triphosphates (ATP, CTP, UTP

and GTP)

- RNA polymerase moves stepwise along the DNA, unwinding the DNA helix just ahead to

expose a new region of the template strand for complementary base-pairing. The growing

RNA chain is extended by one nucleotide at a time in the 5’-3’ direction.

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Several types of RNA are produced in cells

Type of RNA Function

Messenger RNA(mRNA)

Code for proteins

Ribosomal RNA

(rRNA)

Form part of the structure of the ribosome and participate

in protein synthesis

Transfer RNA

(tRNA)

Used in protein synthesis as adaptors between mRNA and

amino acids

Small RNA (snRNA) Used in pre-mRNA splicing and other cellular processes

Small nucleolar RNA

(snoRNA)

Used to process and chemically modify rRNAs

MicroRNA (miRNA) Regulate gene expression typically by blocking

translation of selective mRNAs

Small interfering

RNA (siRNA)

Turn off gene expression by directing degradation of

selective mRNAs and the establishment of compact

chromatin structures

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RNA polymerases come in different forms

- RNA polymerases are made up of multiple subunits (although some phage and

organelles do encode single subunit enzymes that perform the same task)

- Bacteria have only one RNA polymerase

- Eukaryotes have three RNA polymerases: RNA Pol I, RNA Pol II, and RNA Pol III

(Pol II is responsible for protein-coding genes; Pol I transcribes the large ribosomal RNA

precursor gene; Pol III transcribes tRNA genes, some small nuclear genes, and the 5S

rRNA gene.

Prokaryotic Eukaryotic

Bacterial Archaeal RNA Pol I RNA Pol I I RNA Pol I I I

β’ A’/ A’’ RPA1 RPB1 RPC1

β B RPA2 RPB2 RPC2

α’(αI) D RPC5 RPB3 RPC5

α’’(αII) L RPC9 RPB11 RPC9

ω K RPB6 RPB6 RPB6

[+6 others] [+9 others] [+7 others] [+11 others]

The subunits of RNA polymerases

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Comparison of the crystal structures of prokaryotic

and eukaryotic RNA polymerases

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The phases of the transcription cycle:

Initiation, Elogation, and Termination

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The initiation phase of the transcription

- A promoter is the DNA sequence that

initially binds the RNA polymerase.

- The promoter-polymerase complex

undergoes structural changes required for

initiation to proceed.

- The DNA around the point wheretranscription will start unwinds, and the

base pairs are disrupted, producing a

“bubble” of single-stranded DNA.

- Transcription always occurs in a 5’ to 3’

direction. That is, the new ribonucleotide

is added to the 3’-end of the growingchain.

- Once the RNA polymerase has

synthesized a short stretch of RNA

(approximately 10 bases), it shifts into the

elongation phase

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The elongation and termination phase of the transcription

Elongation:

- This transition requires furtherconformational changes in polymerase

that lead to grip the template more firmly.

- RNA polymerase performs an

impressive range of tasks in addition to

the catalysis of RNA synthesis. It unwinds

the DNA in front and re-anneals it behind;it dissociates the growing RNA chain from

the template as it moves along; and it

performs proofreading functions.

Termination:Once the RNA polymerase has transcribed

the length of the gene (or genes), it must

stop and release the RNA product. In

some cells, there are specific, well-

characterized, sequences that trigger

termination.

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The transcription cycle in

bacteria

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Features of bacterial promoters

- There are two conserved sequences: -35 and -10 regions (or elements), each of six

nucleotides, are separated by a nonspecific stretch of 17-19 nucleotides.- An addition DNA element that binds RNA polymerase is found in some strong promoter, for

example those directing expression of the rRNA genes. This is called UP-element and

increase polymerase binding by providing an additional specific interaction between the

enzyme and the DNA

- Some promoters lack a -35 region and instead has a so-called “extended -10 element”. This

comprises a standard -10 region with an additional short sequence element at its upstream end.

TTGACA TATAATStart site

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The sigma (σ) factor mediates binding of polymerase

to the promoter

- Core enzyme: Bacterial RNA polymerase without the sigma (recognition subunit)

- Sigma factor: A subunit of bacterial RNA polymerase that recognizes and binds to the promoter sequence

- The sigma factor can be divided into four regions: σ1, σ2, σ3 and σ4. The regions that

recognize the -10 and -35 elements of promoter are σ2 and σ4, respectively

- The “extended -10 element” is recognized by σ3

- The UP-element of promoter is not recognized by sigma factor, but is instead recognized by a

carboxyl terminal domain of the α subunit, called αCTD

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RNA polymerase transcribes a bacterial gene

- Sigma factor recruit RNA polymerase

core enzyme to the promoter.-Transcription is initiated by RNA

polymerase without the need for a

primer

- Once the RNA polymerase has

synthesized approximately 10

nucleotide of RNA, the sigma factor is

released, enabling the polymerase tomove forward and continue transcribing

without it.

- Chain elongation then continue until

the enzyme encounters the signal of

terminator in the DNA, where

polymerase halts and releases both

DNA template and newly made RNA

chain

- After the polymerase is released at a

terminator, it re-associates with sigma

factor and searches for a promoter,

where it can begin the process of

transcription again.

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Both strands of DNA can be used as template

The direction of transcription is determined by the orientation of thepromoter at the beginning of each gene

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Transcriptional terminator

Sequences called terminators trigger the elongating polymerase to

dissociate from the DNA and release the RNA chain it has made.

Rho-dependent terminator:Transcriptional terminator requires

a protein called Rho.

Rho (ρ) protein: Protein factor

needed for successful termination

at certain transcriptionalterminators

Rho-independent terminator:(intrinsic terminator)

Transcriptional terminator that does

not need Rho protein

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Rho-dependent terminator

- Rho protein: a ring-shape protein with six identical subunits, binds to single-strandedRNA as it exits the polymerase.

- Rho protein also has an ATPase activity: once attached to the transcript, it use the

energy derived from ATP hydrolysis to wrest the RNA from the template and from

polymease.

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Rho-independent terminator (intrinsic terminator)

- Rho-independent terminators consist of two sequence elements:a short inverted repeats (of about

20 nucleotides) followed by a stretch of about 8 A:T base pairs.

- When polymerase transcribes an inverted sequence, the resulting RNA can form a stem-loop

structure (hairpin). The hairpin is believed to cause termination by disrupting the elongation

complex.

- The hairpin only work as an efficient terminator when it is followed by a stretch of A:U base pair.

At the time the hairpin forms, the growing RNA chain will be held on the template at the active site

by only A:U base pairs. As A:U base pairs are the weakest of all base pairs, they are more easilydisrupted by the effects of the hairpin, and so the RNA will more readily dissociate.

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A model for how the Rho-independent terminator might work

a. The hairpin forms in the RNA as

soon as that region has been

transcribed by RNA polymerase

b. That RNA structure disrupted RNA polymerase just as the enzyme is

transcribing the AT rich stretch od

DNA downstream

c. The combination of the hairpinstructure and the weak interaction

between the stretch Us in the RNA and

As in the template conspire to pull the

transcript from the template,

terminating further elongation.

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Transcription in eukaryotes

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Species Gene density

(genes/ Mb)

Average number of

introns per gene

Percentage of DNA

that is repetitive

PROKARYOTES (bacteria)

Escher ichia coli 950 0

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Most human genes are broken into exons and introns

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Transcription in prokaryotes and eukaryotes

Prokaryotes Eukaryotes

- Bacteria have only one RNA

polymerase

-Eukaryotes have three RNA polymerases:

RNA Pol I, RNA Pol II, and RNA Pol III (Pol

II is responsible for protein-coding genes; Pol

I transcribes the large ribosomal RNA

precursor gene; Pol III transcribes tRNAgenes, some small nuclear genes, and the 5S

rRNA gene.

- Bacteria require only one

additional initiation factor

(sigma factor) that mediates

binding of polymerase to the promoter

- Several initiation factors are required for

efficient and promoter-specific initiation in

eukaryotes. These are called the general

transcription factors (GTFs)- Rather, additional factors are required,

including the so-called Mediator complex,

DNA-binding regulatory proteins, and

chromatin-modifying enzymes

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RNA polymerase II core promoter

- BRE: TFIIB recognition element

- TATA box

- Inr: Initiator element- DPE: downstream promoter element

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RNA polymerase I promoter region

- The promoter for the rRNA gene comprise two part: the core element

and the UCE (Upstream Control Element)- In addition to Pol I, initiation requires two other factors, called SL1 and

UBF. SL1 comprises TBP and three TAFs specific for Pol I transcription.

SL1 binds DNA only in presence of UBF.

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RNA polymerase III promoter region

- Pol III promoter come in various forms. Some Pol III (for tRNA genes)

consist of Box A and Box B; other contain Box A and Box C (for 5S rRNA

gene); and still others contain a TATA element like those of Pol II.

-The factors called TFIIIB and TFIIIC are required for the transcription of

tRNA genes, and those plus TFIIIA for the 5S rRNA gene.

RNA l II f i iti ti l ith

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RNA polymerase II forms a pre-initiation complex with

GTFs at the promoter

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General transcription factors associated with

RNA polymerase II

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I n vivo , transcription initiation requires additional proteins

- One reason for these additional requirements is that the DNA template in vivo is packaged intonucleosomes and chromatin. This condition complicates binding to the promoter of polymerase and

its associated factor.

- Transcriptional regulatory proteins called activators help recruit polymerase to the promoter,

stabilizing its binding there

- Mediator complex is associated with the CTD “tail” of the large polymerase subunit through one

surface, while presenting other surfaces for interaction with the DNA-bound activators

*** the role of mediator will be discussed in next chapter (gene regulation)

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RNA processing:

RNA capping, Splicing, and Polyadenylation

- Processing events include the following: capping the 5’-end of the RNA; Splicing

(non-coding introns are removed from RNA to generate the mature mRNA); and

Polyadenylation of the 3’-end of the RNA

- RNA processing enzymes are recruited by the tail of the polymerase.

- The CDT tail contains a series of repeat of the hepapeptide sequence: Tyr-Ser-Pro-Thr-

Ser-Pro-Ser. Phosphorylation of Ser at position 5 is associated with recruitment of

capping factors. Phosphorylation of Ser at position 2 is associated with recruitment of

splicing factors

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RNA processing: RNA capping

RNA capping involves the addition of

a modified guanine base to the 5’end

of RNA. The 5’ cap is created in three

enzymatic steps:

-The γ-phosphate at the 5’ens of the

RNA is removed by an enzyme called

RNA triphosphatase

- The enzyme guanylyl transferasecatalyzes the nucleophilic attack of

the resulting terminal β-phosphate on

the α-phosphoryl group of a molecule

of GTP.

- The newly added guanine and the purin at the original 5’end of the

mRNA are further modified by the

addition of methyl groups by enzyme

methyl transferase

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RNA processing: RNA capping

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Introns are removed by RNA splicing

- Special nucleotide sequences signal the beginning and the end of an intron. The special

sequences are recognized by small nuclear ribonucleoproteins (snRNPs) which cleave

the RNA at the intron-exon borders and covalently link the exons together.

-A, G, U and C are standard RNA nucleotides

-R stands for either A or G; Y stands for either C or U

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The splicing reaction

- A particular adenine nucleotide in

the intron sequence attacks the 5’ splice site and cuts the sugar-

phosphate backbone of the RNA at the

point

- the cut 5’end of the intron becomes

covalently linked to the adeninenucleotide to form a branched

structure.

- The free 3’-OH end of the exon

sequences then reacts with the start of

next exon sequence, joining the twoexons together into continuous coding

sequence and release the intron in the

form of a lariat.

- The lariat containing the intron is

eventually degraded.

Th li i ti d t il f th t t f

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The splicing reaction: details of the structure of

the lariat branch

The cut 5’end of the intron islinked to the 2’-OH group of the

ribose of the branchpoint adenine

nucleotide

RNA splicing is catalyzed by an assembly of snRNPs

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plus other proteins, which form the spliceosome

- The branchpoint site (A) is firstrecognized by the BBP (branch-point-

binding protein) and U2AF, a helper

protein.

- The U2 snRNP displaces BBP and U2AF

and forms base pairs with the branch-point

site consensus sequence- U1 snRNA forms base pairs with the 5’

splice junction

- The U4/U6.U5 “triple” snRNP enters the

spliceosome. In this “triple”, the U4 and

U6 snRNPs are held firmly together by

base-pair interactions and the U5 snRNPis more loosely associated

- Several RNA-RNA rearrangements the

occur that break apart the U4/U6 base

pairs and allow the U6 snRNP to displace

U1 at the 5’ splice juction.

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plus other proteins, which form the spliceosome

(continued)

Subsequent rearrangements create the

active site of the spliceosome and

position the appropriate portions of the

pre-mRNA substrate for the splicing tooccur.

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Three classes of RNA splicing

Group I and Group II introns: splice themselves out of pre mRNA without the need for

the spliceosome. They are called self-splicing introns

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Self-splicing introns

Si l d lti l d t b

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Single genes can produces multiple products by

alternative splicing

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RNA processing: Polyadenylation

- Two proteins complexes are carried

by the CTD of polymerase as it

approaches to the end of gene: CPSF

(Cleavage and Polyadenylation

Specificity Factor) and CstF (Cleavage

stimulation Factor).- The sequences which, once

transcribed into RNA, trigger transfer

of CPSF and CstF are called poly-A

signal. Once these factors are bound to

the RNA, other proteins are recruited

as well, leading to RNA cleavage andthen polyadenylation.

- Polyadenylation is mediated by an

enzyme called poly-A polymerase,

which add about 200 adenines to the

RNA’s 3’ end produced by cleavage

RNA i P l d l i

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RNA processing: Polyadenylation

(continued)

- Poly-A polymerase use ATP as a

precursor and adds the nucleotides using

the same chemistry as RNA polymerase

- It is not clear what determines the length

of the poly-A tail, but that processinvolves other proteins that bind

specifically to the poly-A sequence.

- The polymerase then dissociates from

the template, releasing the mature RNA

- The mature RNA is then transported

from the nucleus.

Transport of mRNAs out of the nucleus

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Transport of mRNAs out of the nucleus

-A typical mature mRNA carries a collection of proteins that identifies it as being mRNAdestined for transport.

- Export takes place through a special structure in the nuclear membane called the nuclear

pore complex.

- Once in the cytoplasma, the proteins are discarded, and are then recognized to import

back to the nucleus where they associate with another mRNA and repaet the cycle.

Prokaryotes and eukaryotes handle their transcripts

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somewhat differently (1)


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molbiol 07

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