Mechanisms of Transcription

The flow of genetic information

Gene: The region of

controls a discrete
characteristic of an
usually corresponding
protein or RNA.

DNA that
hereditary
organism,
to a single

Genome size is related to the complexity of the organism

Species

Genome size (Mb)

Approximate
number of genes

Gene density
(genes/ Mb)

Mycoplasma genitalium

0.58

500

860

Streptococus pneumonia

2.2

2,300

1,600

Escherichia coli

4.6

4,400

950

Fungi: Saccharomyces cerevisiae

12

5,800

480

Invertebrates: Caenorhabditis elegans

97

19,000

200

Invertebrates: Drosophila melanogaster

180

13,700

80

Vertebrate: Homo sapiens

2,900

27,000

9.3

Vertebrate: Mus musculus

2,500

29,000

12

Plants: Arabidopsis thaliana

125

25,500

200

Plants: Oryza sativa (rice)

430

>45,000

>100

PROKARYOTES (bacteria)

EUKARYOTES

* 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

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 proteincoding regions. These interspersed non-protein-encoding regions is called
introns.

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.

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

The chemical structure of RNA differs slightly from that of DNA

Transcription produces RNA complementary to one strand of DNA

Uracil forms base pair with Adenine

The enzymes that carry out transcription are called

RNA polymerase

- RNA polymerases catalyze the formation of the phosphodiester bonds that link the
nucleotides 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.

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 Used to process and chemically modify rRNAs
(snoRNA)
MicroRNA (miRNA) Regulate gene expression typically
translation of selective mRNAs
Small interfering
RNA (siRNA)

by

blocking

Turn off gene expression by directing degradation of

selective mRNAs and the establishment of compact
chromatin structures

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.

The subunits of RNA polymerases

Prokaryotic
Bacterial

Eukaryotic

Archaeal

RNA Pol I

RNA Pol II

RNA Pol III

A/ A

RPA1

RPB1

RPC1

RPA2

RPB2

RPC2

(I)

RPC5

RPB3

RPC5

(II)

RPC9

RPB11

RPC9

RPB6

RPB6

RPB6

[+6 others]

[+9 others]

[+7 others]

[+11 others]

Comparison of the crystal structures of prokaryotic

and eukaryotic RNA polymerases

The phases of the transcription cycle:

Initiation, Elogation, and Termination

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 where
transcription 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 growing
chain.
- Once the RNA polymerase has
synthesized a short stretch of RNA
(approximately 10 bases), it shifts into the
elongation phase

The elongation and termination phase of the transcription

Elongation:
- This
transition
requires
further
conformational 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, wellcharacterized, sequences that trigger
termination.

The transcription cycle in

bacteria

Features of bacterial promoters

TTGACA

TATAAT

Start site

- 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.

The sigma () 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: 1, 2, 3 and 4. The regions that
recognize the -10 and -35 elements of promoter are 2 and 4, respectively
- The extended -10 element is recognized by 3
- The UP-element of promoter is not recognized by sigma factor, but is instead recognized by a
carboxyl terminal domain of the subunit, called CTD

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 to
move 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.

Both strands of DNA can be used as template

The direction of transcription is determined by the orientation of the

promoter at the beginning of each gene

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 () protein: Protein factor
needed for successful termination
at
certain
transcriptional
terminators

Rho-independent terminator:
(intrinsic terminator)
Transcriptional terminator that does
not need Rho protein

Rho-dependent terminator

- Rho protein: a ring-shape protein with six identical subunits, binds to single-stranded
RNA 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.

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 easily
disrupted by the effects of the hairpin, and so the RNA will more readily dissociate.

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 hairpin

structure 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.

Transcription in eukaryotes

Eukaryotic genes are interrupted by noncoding sequences

Species

Gene density
(genes/ Mb)

Average number of
introns per gene

Percentage of DNA
that is repetitive

950

<1

Fungi: Saccharomyces cerevisiae

480

0.04

3.4

Invertebrates: Caenorhabditis elegans

200

6.3

Invertebrates: Drosophila melanogaster

80

12

Vertebrate: Homo sapiens

9.3

46

PROKARYOTES (bacteria)
Escherichia coli
EUKARYOTES

Most human genes are broken into exons and introns

Transcription in prokaryotes and eukaryotes

Prokaryotes

Eukaryotes

- Bacteria have only one RNA -Eukaryotes have three RNA polymerases:
polymerase
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.
- 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

RNA polymerase II core promoter

- BRE: TFIIB recognition element

- TATA box
- Inr: Initiator element
- DPE: downstream promoter element

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.

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 polymerase II forms a pre-initiation complex with

GTFs at the promoter

General transcription factors associated with

RNA polymerase II

In vivo, transcription initiation requires additional proteins

- One reason for these additional requirements is that the DNA template in vivo is packaged into
nucleosomes 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)

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-ThrSer-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

RNA processing: RNA capping

RNA capping involves the addition of
a modified guanine base to the 5end
of RNA. The 5 cap is created in three
enzymatic steps:
-The -phosphate at the 5ens of the
RNA is removed by an enzyme called
RNA triphosphatase
- The enzyme guanylyl transferase
catalyzes the nucleophilic attack of
the resulting terminal -phosphate on
the -phosphoryl group of a molecule
of GTP.
- The newly added guanine and the
purin at the original 5end of the
mRNA are further modified by the
addition of methyl groups by enzyme
methyl transferase

RNA processing: RNA capping

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

The splicing reaction

- A particular adenine nucleotide in
the intron sequence attacks the 5
splice site and cuts the sugarphosphate backbone of the RNA at the
point
- the cut 5end of the intron becomes
covalently linked to the adenine
nucleotide 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 two
exons together into continuous coding
sequence and release the intron in the
form of a lariat.
- The lariat containing the intron is
eventually degraded.

The splicing reaction: details of the structure of

the lariat branch

The cut 5end of the intron is

linked to the 2-OH group of the
ribose of the branchpoint adenine
nucleotide

RNA splicing is catalyzed by an assembly of snRNPs

plus other proteins, which form the spliceosome
- The branchpoint site (A) is first
recognized by the BBP (branch-pointbinding 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 snRNP
is 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.

RNA splicing is catalyzed by an assembly of snRNPs

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 to
occur.

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

Self-splicing introns

Single genes can produces multiple products by

alternative splicing

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 and
then polyadenylation.
- Polyadenylation is mediated by an
enzyme called poly-A polymerase,
which add about 200 adenines to the
RNAs 3 end produced by cleavage

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 process
involves 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

-A typical mature mRNA carries a collection of proteins that identifies it as being mRNA
destined 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

somewhat differently (1)

Prokaryotes and eukaryotes handle their transcripts

somewhat differently (2)

Prokaryotes and eukaryotes handle their transcripts

somewhat differently (3)