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RNA and transcripti on

Click to edit Master subtitle Jesil Mathew. A style

RNA

Differences between RNA and DNA

Mainly seen in cytoplasm bases sugar by alkali

100-5000 Ribose Uracil Degraded

Types
mRNA rRNA
28S, Very

2-5% of total RNA, degraded quickly

About 80% of all rna in the cell

18S and 5S are the majour variants, stable, involved in the protein biosynthesis

tRNA
15% Very

About 60 different species present.

of the total RNA in the cell. stable

sRNA

cell

(small RNA)- 1-2% of total RNA in the

Small

Nuclear RNAs (snRNAs)are a subgroup of small RNA. (Important subgroups are U1, U2, U3,

Transcription
Synthesis

of RNA from DNA template using

RNA polymerase,
Four

steps: Binding of RNA polymerase to the

template, Initiation, Chain Elongation and Termination.

RNA

is synthesized complementary to Non

coding strand (antisense strand or template stand) of DNA.

Precursors

of

RNA

synthesis

are

four

Basic features of RNA synthesis

A

polymerisation reaction in which a 3OH

group of one nucleotide reacts with 5 triphosphate of the second nucleotide releasing a pyrophosphate and forming a phosphodiester bond. (Similar to DNA synthesis).
The

RNA chain grows in the 5

direction
RNA

polymerase is able to initiate the chain

RNA polymerase
E.

coli RNA Polymerase:

of five sub groups

Consists

Two identical subunits each of types , and

One One

of the largest enzyme known with a total molecular wt. of 465,000 subunit dissociates during elongation stage of RNA polymerisation. term core enzyme is used to describe - free unit. (2) complete enzyme 2 is called the holoenzyme.

The The The

Assignment
Explain

the following techniques

protection method sulphate protection method

DNAse Foot

printing

Dimethyl

Transcription
Genes

are composed of a number of distinct regions, which control and encode the desired product. regions are generally of the form promoter -- gene(s) -- terminator, as shown below. of gene expression by the promoter region avoids the synthesis of unnecessary products, although control of expression following initiation may also take place. on regulatory pressures, the

These

Control

Depending

Downstream and upstream

The

first base transcribed was chosen as a

reference point and numbered +1.

The

direction

of

transcription

was

called

downstream.
All

upstream bases which are not transcribed

were given negative numbering starting from reference.

The promoter
The

site of binding of RNA polymerase on DNA

molecule is known as promoter.

These

sequences occur upstream from the

transcription start site (+1) at positions -10 and -35.

The

-35 region affects the binding of the RNA the Pribnow box) subsequent

polymerase, whilst the -10 region (originally called transcription.

Pribnow box
In

a region 5-10 bases upstream to the start

+1, there exist a consensus sequence. These sequences are known as Pribnow box.
When

there is no relation between there

sequences, one would expect each base to occur at each position 25% of the time.
That

is in a six base sequence, the possibility

of a base to occur will be 25% i.e., (A/T/C/G)(A/T/C/G)(A/T/C/G)(A/T/C/G)

Pribnow box
Examination

of more than 100 E coli promoters

have shown that the frequency of occurrence of the bases is T80 A95 T45 A60 A50 T95
Pribnow

box

is

thought

to

orient

RNA

polymerase, and to be the region at which the double helix opens to form the open promoter complex.
The

Pribnow box or Pribnow-Schaller box

-35 sequence
The

sequence at -35 (the -35 element) usually

consists of the six nucleotides TTGACA. Its presence allows a very high transcription rate
Probability

of occurrence of each nucleotide for

-35 sequence is T69 T 79G61 A56 C54 A54.

It

should be noted that the above promoter

sequences are only recognized by the sigma70 protein that interacts with the prokaryotic

Consensus and its importance

A

couple of important points exist about the consensus. First, not all bases in the consensus are conserved to the same amount. The bases marked with bold type and underlined are more conserved than the others, and the -10 region is more conserved overall than is the -35 region. Secondly, the promoter sequence is asymmetrical; that is, it reads differently in one direction than in the other. TTGACA (Compare this to the recognition sequence for the restriction enzyme BamHI, GGATCC.) This asymmetry means that RNA polymerase

Initiation
Binding

of RNA polymerase to dsDNA in the promoter region. Promoters are specific sequences present at the start of the genes, that is to the 5-side (upstream) of the coding region. RNA polymerase binds to one of several specificity factors, , 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

Initiation
RNA

polymerase can exist in one of two states

The The

initiation state appropriate for the tight binding to promoters (closed promoter complex). elongation state for loose binding and mRNA synthesis (open promoter complex).

must be present for tight binding to occur, although once bound it will dissociate, leaving RNA polymerase to transcribe the gene.

Closed and open promoter complexes

Weak promoters

Weak promoters are those promoters were the recognition and binding by RNA polymerase is less strong.

In a given time the number of RNA molecules synthesized from genes with weak promoters is much less than from a strong promoter with the result that fewer mRNA molecules are made per unit time genes of weak promoters.

Promoter strength is one factor that determines the number of copies of each protein molecule present in the cell.

The difference between weak and strong promoter lies in the sequences of the -35 and -10 regions.

Auxiliary proteins
Some

bacterial promoters require an activator protein for effective initiation.

Two

Example-1

phage promoters pI and pre, both of which are inactive unless an auxillary protein , the encoded cII protein is present.

Example
E

II

coli lac promoter includes a -35 sequence but does not bind RNA polymerase significantly unless the cyclic AMP (cAMP) receptor protein (CRP) is also bound.

Initiation conted
Promoters

can differ in "strength"; that is, how

actively they promote transcription of their adjacent DNA sequence.

Promoter

strength is in many (but not all) matter of how tightly RNA

cases,

polymerase and its associated accessory proteins bind to their respective DNA sequences.

 Additional

transcription regulation comes

from transcription factors that can affect the stability of the holoenzyme structure at initiation.
Most

transcripts originate using adenosine-5'-

triphosphate (ATP) and, to a lesser extent, guanosine-5'-triphosphate (GTP) (purine

nucleoside triphosphates) at the +1 site.

Initiation conted
The

initiating nucleoside triphosphate binds to

the enzyme in the open promoter complex and forms a hydrogen bond with the complementary DNA base.
The

elongation site (Catalytic site) is then filled

with a nucleoside triphosphate that is selected by its ability to hydrogen bond with the next base in the DNA strand.
The

two nucleotides are then joined together,

Initiation conted..
The

polymerisation of first few nucleotides is from the process that occurs

different

henceforth. The main difference is that the strict one-by-one base template reading is not yet established.
During

this initial period short oligonucleotides

are often rleased from DNA indicating that RNA polymerase stops and restarts at the initiation site.

Chain elongation
After

several nucleotides ate added to the growing chain (mostly upto eight), RNA polymerase undergoes a conformational change and loses its subunit. marks the transition form stuttering of the initiation phase as described before. most of the elongation is carried out by core enzyme. core enzyme moves along with the DNA, binding a nucleoside to pair with the next DNA base and opening the DNA helix as it moves. DNA helix recloses as synthesis proceeds.

This

Thereafter The

The

Chain elongation - Pause

Chain

elongation process is not occurring at a constant rate. will be reduction in rate when particular regions of DNA are passed, then continues at the normal rate. This reduction of rate is known as a pause. of pauses along stretches of DNA of known sequences shows that pausing frequently causes sequences that form hairpins in the RNA. at least half of the pause sites have no recognizable features.

There

Analysis

But

Termination
Two

termination mechanisms are well known:

Intrinsic

termination (also called Rhoindependent transcription termination) termination uses a termination factor called factor(rho factor) which is a protein to stop RNA synthesis at specific sites.

Rho-dependent

Intrinsic termination (Rhoindependent transcription termination)

Involves The

terminator sequences within the RNA that signal the RNA polymerase to stop. terminator sequence is usually a palindromic sequence that forms a stemloop hairpin structure that leads to the dissociation of the RNAP from the DNA template. i.e., intrastrand base pairing and formation of cruciform structure. the loop end of the putative stem, there is high G+C sequence. RNA polymerase usually slows down when synthesising the corresponding RNA segment.

Near

 Intrinsic

termination (also called Rhoindependent transcription termination) involves terminator sequences within the RNA that signal the RNA polymerase to stop. The terminator sequence is usually a palindromic sequence that forms a stemloop hairpin structure that leads to the dissociation of the RNAP from the DNA template. termination uses a termination factor called factor(rho factor) which is a protein to stop RNA synthesis at specific sites. This protein binds at a rho utilization site on the nascent RNA strand and runs along the mRNA towards the RNAP. A

Rho-dependent