Q1) what is role of enhancers and promoters in transcription of eukaryotes?

ANS:

Enhancer
1) an enhancer is a short region of DNA that can be bound with proteins (namely, the trans-acting
factors, much like a set of transcription factors) to enhance transcription levels of genes (hence the name)
in a gene cluster. While enhancers are usually cis-acting, an enhancer does not need to be particularly
close to the genes it acts on, and sometimes need not be located on the same chromosome.

2) Like bacteria, eukaryotes use gene regulatory proteins (activates and repressors) to regulate the
expression of their genes but in somewhat different way. The DNA sites to witch the eukaryote gene
activates bound were originally termed enhancers, since their presence “enhanced”, or increased, the rate
of transcription dramatically. It came as a surprise when, in 1979, in was discovered that those activator
proteins could be bound thousands of nucleotide pairs away from promoter.

3) Enhancer is the DNA element that, upon binding with transcription factors
(activators), can enhance transcription. It may be located either upstream or
downstream of the transcriptional initiation site.  However, most of them are located
upstream.  In prokaryotes, enhancers are quite close to the promoter, but eukaryotic
enhancers could be far from the promoter.

(a) E. coli glnA gene

(b) Yeast GAL1 and GAL10 genes
 (c) Human b globin gene cluster

4) Location of enhancer- In eukaryotic cells the structure of the chromatin complex of DNA is folded in a way that
functionally mimics the supercoiled state characteristic of prokaryotic DNA, so that although the enhancer DNA is far from the
gene in regard to the number of nucleotides, it is geometrically close to the promoter and gene. This allows it to interact with
the general transcription factors and RNA polymerase II. An enhancer may be located upstream or downstream of the gene that it
regulates. Furthermore, an enhancer does not need to be located near to the transcription initiation site to affect the transcription
of a gene, as some have been found to bind several hundred thousand base pairs upstream or downstream of the start site.
Enhancers do not act on the promoter region itself, but are bound by activator proteins. These activator proteins interact with
the mediator complex, which recruits polymerase II and the general transcription factors which then begin transcribing the genes.
Enhancers can also be found within introns. An enhancer's orientation may even be reversed without affecting its function.
Additionally, an enhancer may be excised and inserted elsewhere in the chromosome, and still affect gene transcription. That is
one reason that intron polymorphisms may have effects although they are not translated.

Promotor:
1) A promoter is a regulatory region of DNA located upstream (towards the 5' region) of of a gene, providing a control point for regulated
gene transcription.

The promoter contains specific DNA sequences that are recognized by proteins known as transcription factors. These factors bind to the promoter
sequences, recruiting RNA polymerase, the enzyme that synthesizes the RNA from the coding region of the gene.
2) PROMOTER ELEMENTS

A. Core promoter - the minimal portion of the promoter required to properly initiate transcription

 Transcription Start Site (TSS)

 Approximately -34
 A binding site for RNA polymerase
 General transcription factor binding sites

B. Proximal promoter - the proximal sequence upstream of the gene that tends to contain primary regulatory elements

 Approximately -250
 Specific transcription factor binding sites

3) Difference between Eukaryotic and Prokaryotic Promoters

A. Prokaryotic promoters

In prokaryotes, the promoter consists of two short sequences at -10 and -35 positions upstream from the transcription start site.

 The sequence at -10 is called the Pribnow box, or the -10 element, and usually consists of the six nucleotides TATAAT. The Pribnow
box is absolutely essential to start transcription in prokaryotes.
 The other sequence at -35 (the -35 element) usually consists of the six nucleotides TTGACA. Its presence allows a very high
transcription rate.

B. Eukaryotic promoters

Eukaryotic promoters are extremely diverse and are difficult to characterize. They typically lie upstream of the gene and can have regulatory
elements several kilobases away from the transcriptional start site. In eukaryotes, the transcriptional complex can cause the DNA to bend back on
itself, which allows for placement of regulatory sequences far from the actual site of transcription. Many eukaryotic promoters, contain a TATA
box (sequence TATAAA), which in turn binds a TATA binding protein which assists in the formation of the RNA polymerase transcriptional
complex. The TATA box typically lies very close to the transcriptional start site (often within 50 bases).
Q2) Differentiate between the initiation of transcription in eukaryote and prokaryotes?

In Eukaryotes:

While very similar to that in prokaryotes, the "machinery" and control sequences of
transcription in eukaryotes is much more complex, and there are numerous RNA
polymerases.

Among eukaryotes that regulate the transcription of individual genes, the core promoter of protein-
encoding gene contains binding sites for the basal transcription complex and RNA polymerase II, and is
normally within about 50 bases upstream of the transcription initiation site. Further transcriptional
regulation is provided byupstream control elements (UCEs), usually present within about 200 bases
upstream of the initiation site. The core promoter for Pol II sometimes contains a TATA box, the highly
conserved DNA recognition sequence for the TATA box binding protein, TBP, whose binding initiates
transcription complex assembly at the promoter.

Some genes also have enhancer elements that can be thousands of bases upstream or downstream of the
transcription initiation site. Combinations of these upstream control elements and enhancers regulate and
amplify the formation of the basal transcription complex.

In eukaryotes, there are three classes of RNA polymerases: I, II and III.  This section will focus on the
RNA polymerase II (Pol II), which is involved in the transcription of all protein genes.  Transcription
by RNA Pol I and Pol III .
 Structure of the human TBP core domain complexed with DNA as determined by x-ray
crystallography.  The DNA includes the TATA element.  PDB ID = 1CDW.

Initiation

RNA Pol II does not contain a subunit similar to the prokaryotic s factor, which can recognize the
promoter and unwind the DNA double helix.  In eukaryotes, these two functions are carried out by a
set of proteins called general transcription factors.  The RNA Pol II is associated with six general
transcription factors, designated as TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH, where "TF" stands
for "transcription factor" and "II" for the RNA Pol II.

TFIID consists of TBP (TATA-box binding protein) and TAFs (TBP associated factors).  The role of

TBP is to bind the core promoter (Figure 4-E-1).  TAFs may assist TBP in this process.  In human
cells, TAFs are formed by 12 subunits.  One of them, TAF250 (with molecular weight 250 kD), has
the histone acetyltransferase activity, which can relieve the binding between DNA and histones in the
nucleosome.

In Prokaryotes:

The most studied RNA polymerase is that from E.coli, so we will study it as the
prototype of the RNA polymerases. The holoenzyme is a 449 kD protein composed of
a "core enzyme" and a "-subunit", and the entire complex is denoted  
(core)The core enzyme directs the polymerization reaction, and it has 4
subunits:  core enzyme = 'he inorganic ions Zn2+ (two of them in
the ' subunit) and Mg2+ are required for catalytic activity and the three-dimensional
structure of the enzyme resembles a hand. The thumb of the hand can be envsioned as
grasping a piece of B DNA that lies in a channel represented by the curved fingers and
palm of the hand. This channel is cylindrical, with dimensions on the order of 25 A by
55 A. These dimensions allow a fit of about 16 base pairs of B DNA.

2) The following steps occur, in order, for transcription initiation:

 RNA polymerase (RNAP) binds to one of several specificity factors, σ, to form a holoenzyme. In this form, it can

recognize and bind to specific promoter regions in the DNA. The -35 region and the -10 ("Pribnow box") region comprise

the basic prokaryotic promoter, and |T| stands for the terminator. The DNA on the template strand between the +1 site and
 the terminator is transcribed into RNA, which is then translated into protein.At this stage, the DNA is double-stranded

("closed"). This holoenzyme/wound-DNA structure is referred to as the closed complex.

 The DNA is unwound and becomes single-stranded ("open") in the vicinity of the initiation site (defined as +1). This

holoenzyme/unwound-DNA structure is called the open complex.

 The RNA polymerase transcribes the DNA (the beta subunit initiates the synthesis), but produces about 10 abortive

(short, non-productive) transcripts which are unable to leave the RNA polymerase because the exit channel is blocked by the

σ-factor.

 The σ-factor eventually dissociates from the holoenzyme, and elongation proceeds.
Transcription - Initiation

This depicts the initiation of transcription. RNAP stands for the enzyme RNA polymerase.
Forluvoft, Wikipedia Commons

The initiation of transcription in bacteria begins with the binding of RNA polymerase to the promoter in DNA.
Transcription initiation is more complex in eukaryotes, where a group of proteins called transcription factors
mediate the binding of RNA polymerase and the initiation of transcription.

Q3) What are the factors controlling termination of transcription in prokaryotes?

In prokaryotes, the transcription is terminated by two major mechanisms: Rho-

independent (intrinsic) and Rho-dependent.

The Rho-Independent termination signals

 The intrinsic terminator sequence is an inverted repeat of GC-rich sequence

followed by 4 or more adenines. The transcribed RNA forms stem-loop
structure at inverted repeats via internal base pairing
  The formation of this stem-loop structure (Fig. 11.1) disrupts hydrogen bonding
between RNA uracils and DNA adenines at site of transcription (weak because
only 2 H-bonds between A and U as compared to 3 between G and C)
 As a result, RNA is released from the DNA template

The Rho-independent termination signal is a stretch of 30-40 bp sequence,

consisting of many GC residues followed by a series of T ( "U" in the transcribed
RNA).  The resulting RNA transcript will form a stem-loop structure to terminate
transcription.

Figure 4-D-2.  The stem-loop structure of the RNA transcript as a termination signal

for the transcription of the trp operon.

Rho-dependent mechanism

Rho is a ~ 50 kD protein, involved in bout half of E. coli transcriptional

terminations.  It has been well established that six Rho proteins form a hexamer to
terminate transcription, but the precise mechanism is not clear.  Experiments
suggest that two components are essential: (i) the upstream Rho loading site and (ii)
the downstream termination site.  The Rho hexamer first binds to the RNA
transcript at the upstream site which is 70-80 nucleotides long and rich in C
residues.  Upon binding, the Rho hexamer moves along the RNA in the 3'
direction.  If movement of the polymerase is slow, the Rho hexamer will catch up
and terminate the transcription at the downstream termination site.  Rho has ATPase
activity which can induce release of the polymerase from DNA.

 Rho-dependent Termination Signals

 Some termination sequences lack the series of adenines which are transcribed
in to URACILS on the RNA. The RNA in such situations needs assistance from
a specific protein (termedRho) which is necessary for termination.
 Rho binds at the 5'end of the RNA and scans down the RNA until it catches up
with an RNA polymerase  which is paused at a stem-loop structure.
 In Rho dependent termination, the Rho protein forces the RNA to separate from
the DNA template.