Rna 2i2sa

4/22/12

RNA and transcripti on Click to edit Master subtitle Jesil Mathew. A style

RNA 

Differences between RNA and DNA 

Mainly seen in cytoplasm

 100-5000  Ribose

bases

sugar

 Uracil  Degraded

by alkali

Types  mRNA  rRNA

– 2-5% of total RNA, degraded quickly

– About 80% of all rna in the cell

 28S,

18S and 5S are the majour variants,

 Very

stable, involved in the protein biosynthesis

 tRNA

– About 60 different species present.

 15%

of the total RNA in the cell.

 Very

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 3’OH

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’

3’

direction  RNA

polymerase is able to initiate the chain

RNA polymerase  E.

coli RNA Polymerase:

 Consists 

of five sub groups

Two identical α subunits

 One

each of types β, β’ and σ

 One

of the largest enzyme known with a total molecular wt. of 465,000 σ subunit dissociates during elongation stage of RNA polymerisation.

 The

term core enzyme is used to describe σ- free unit. (α2ββ’)

 The

complete enzyme α2ββ’σ is called the holoenzyme.

 The

Assignment  Explain

the following techniques

 DNAse  Foot

protection method

printing

 Dimethyl

sulphate protection method

Transcription  Genes

are composed of a number of distinct regions, which control and encode the desired product.

 These

regions are generally of the form promoter -- gene(s) -- terminator, as shown below.

 Control

of gene expression by the promoter region avoids the synthesis of unnecessary products, although control of expression following initiation may also take place.

 Depending

on

regulatory

pressures,

the

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

polymerase, whilst the -10 region (originally called

the

transcription.

Pribnow

box)

subsequent

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, 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

Initiation  RNA

polymerase can exist in one of two states

 The

initiation state appropriate for the tight binding to promoters (closed promoter complex).

 The

elongation state for loose binding and mRNA synthesis (open promoter complex).

 σ-

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.

 Example-1

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

 Two

 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

cases,

a

strength is in many (but not all) matter

of

how

tightly

RNA

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 ed together,

Initiation conted..  The

polymerisation of first few nucleotides is

different

from

the

process

that

occurs

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 σ subunit.

 This

marks the transition form stuttering of the initiation phase as described before.

 Thereafter

most of the elongation is carried out by core enzyme.

 The

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.

 The

DNA helix recloses as synthesis proceeds.

Chain elongation - Pause  Chain

elongation process is not occurring at a constant rate.

 There

will be reduction in rate when particular regions of DNA are ed, then continues at the normal rate. This reduction of rate is known as a pause.

 Analysis

of pauses along stretches of DNA of known sequences shows that pausing frequently causes sequences that form hairpins in the RNA.

 But

at least half of the pause sites have no recognizable features.

Termination  Two

termination mechanisms are well known:

 Intrinsic

termination (also called Rhoindependent transcription termination)

 Rho-dependent

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

Intrinsic termination (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. i.e., intrastrand base pairing and formation of cruciform structure.

 Near

the loop end of the putative stem, there is high G+C sequence. RNA polymerase usually slows down when synthesising the corresponding RNA segment.

 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.

 Rho-dependent

termination uses a termination factor called ρ 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