TERM PAPER PRESENTATION

GP-508

TRANSCRIPTION

The synthesis of RNA molecules using DNA strands as the templates so that the genetic information can be transferred from DNA to RNA.

TRANSCRIPTION

CENTRAL DOGMA OF MOLECULAR BIOLOGY

-FRANCIS CRICK

SIMILARITIES BETWEEN DNA REPLICATION AND TRANSCRIPTION

• Both processes use DNA as the template.

• Phosphodiester bonds are formed in both cases.

• Both synthesis directions are from 5´ to 3´.

replication transcription

template double strands single strand

substrate dNTP NTP

primer yes no

Enzyme DNA polymerase RNA polymerase

product dsDNA ssRNA

base pair A-T, G-C A-U, T-A, G-C

Differences between replication and transcription

“The Protein Players” - RNA polymerases, transcription factors, initiation factors, enhancers, repressors

5'

3'

3'

5'-50 -40 -30 -20 -10 1 10

start -10 region

T A T A A T A T A T T A

(Pribnow box)

-35 region

T T G A C A A A C T G T

Prokaryotic promoter

Consensus sequence

Consensus Sequence

Figure 1-3-2. Transcription of DNA

• The -35 region of TTGACA sequence is the recognition site and the binding site of RNA-pol.

• The -10 region of TATAAT is the region at which a stable complex of DNA and RNA-pol is formed.

Figure 1-3-3. Flow of Genetic Information From DNA to Protein

• RNA polymerase moves along the template strand in the 3' to 5' direction as it synthesizes the RNA product in the 5' to 3' direction using NTPs (ATP, GTP, CTP, UTP) as substrates.

• RNA polymerase does not proofread its work. The RNA product is complementary and antiparallel to the template strand.

• The coding (non-template) strand is not used during transcription. It is identical in sequence to the RNA molecule, except that RNA contains uracil instead of the thymine found in DNA.

• By convention, the base sequence of a gene is given from the coding strand (5' 3').

RNA POLYMERASERNA POLYMERASE

core enzymeholoenzyme

The holoenzyme of RNA-pol in E.coli consists of 5 different subunits: α 2 β β′ ωσ.

ω

β′β

αα

σ

RNA POLYMERASE

subunit MW function

α 36512 Determine the DNA to be transcribed

β 150618 Catalyze polymerization

β′ 155613 Bind & open DNA template

σ 70263Recognize the promoterfor synthesis initiation

RNA-pol of E. Coli

Comparison of Eukaryotic and Prokaryotic RNA Polymerases

STEPS IN TRANSCRIPTION

INITIATIONELONGATIONTERMINATION

• Three phases: initiation, elongation,

and termination.

• The prokaryotic RNA-pol can bind to the DNA template directly in the transcription process.

• The eukaryotic RNA-pol requires co-factors to bind to the DNA template together in the transcription process.

General concepts

• Initiation phase: RNA-pol recognizes the promoter and starts the transcription.

• Elongation phase: the RNA strand is continuously growing.

• Termination phase: the RNA-pol stops synthesis and the nascent RNA is separated from the DNA template.

Transcription of Prokaryotes

a. Initiation

• RNA-pol recognizes the TTGACA region, and slides to the TATAAT region, then opens the DNA duplex.

• The unwound region is about 17±1 bp.

• The first nucleotide on RNA transcript is always purine triphosphate. GTP is more often than ATP.

• The pppGpN-OH structure remains on the RNA transcript until the RNA synthesis is completed.

• The three molecules form a transcription initiation complex.

RNA-pol (α2ββ′σ) - DNA - pppGpN- OH 3′

• No primer is needed for RNA synthesis.

• The σ subunit falls off from the RNA-pol once the first 3′,5′ phosphodiester bond is formed.

• The core enzyme moves along the DNA template to enter the elongation phase.

b. Elongation

• The release of the σ subunit causes the conformational change of the core enzyme. The core enzyme slides on the DNA template toward the 3′ end.

• Free NTPs are added sequentially to the 3′ -OH of the nascent RNA strand.

• RNA-pol, DNA segment of ~40nt and the

nascent RNA form a complex called the transcription bubble.

• The 3′ segment of the nascent RNA hybridizes with the DNA template, and its 5′ end extends out the transcription bubble as the synthesis is processing.

Transcription bubble

c. Termination

• The RNA-pol stops moving on the DNA template. The RNA transcript falls off from the transcription complex.

• The termination occurs in either ρ -dependent or ρ -independent manner.

The termination function of ρ factor

The ρ factor, a hexamer, is a ATPase and a helicase.

ρ-independent termination

• The termination signal is a stretch of 30-40 nucleotides on the RNA transcript, consisting of many GC followed by a series of U.

• The sequence specificity of this nascent RNA transcript will form particular stem-loop structures to terminate the transcription.

RNA

5′TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGGCACCAGCCTTTTT... 3′ DNA

UUUU...…

rplL protein

UUUU...…

5′TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGTCACCAGCCTTTTT... 3′

TRANSCRIPTION IN EUKARYOTES

CHARACTERISTICS OF THE THREE RNA POLYMERASES OF EUKARYOTES

CHARACTERISTICS RNA POL I RNA POL II RNA POL III

Sub units 2+<10 2+<10 2+<10

Target genes Ribosomal RNA genes

m RNA genes t RNA and other small genes

location nucleolus nucleoplasm nucleoplasm

promoters UP of the start point

UP of the start point

DS of the start point

Level of activity Most prominent 50-70 %

20-40% ~10%

Ancillary factors Two ( UBF1, SL1 ) Many ( general, upstream and regulatory)

Three

TF for eukaryotic transcription

Eukaryotic Promoter

Sequence recognized by a transcription factor

Sequence where DNA is denatured determining where transcription starts

Site where other regulatory proteins bind to enhance transcription

A site where regulatory proteins can bind to enhance transcription

TranscripTion EnhancErs and silEncErs

• Both are Binding Sites for Transcription Factors (TF’s)• Enhancers: Increase the amount of Transcription from a

nearby promoter (core + upstream elements)• Silencers: Decrease amount of Transcription from nearby

promoters• Initially Defined as being “Position and orientation

independent”– Found upstream, within, or downstream of genes– Function in either orientation (not always true)

Pre-initiation complex (PIC)

RNA pol II

TF II F

TBP TAFTATA

DNATF II A

TF II B

TF II E

TF II H

• Mechanism of transcription: Involves • Initiation• Elongation• Termination.

Recognizes and binds to TATA box; TBP + 10 TBP associated factors

Binds and stabilizes the TFIID complex

Recruits RNA pol II + TFIIF to the location

Two subunits - RAP38 & RAP74. Rap74 has a helicase activity; RAP38 binds RNAPolII

Two subunits - recruits TFIIH to the complex thereby priming the initiation complex for promoter clearance and elongation complex of 9 subunits. One w/ kinase activity; one w/ helicase activity; one is a cyclin (cdk7)

• Initiation mechanism of transcription:• TFIID recognizes and binds to the TATA box.• TFIIA binds and stabilizes TFIID binding (upstream).• TFІІB bind the promoter downstream of the TFІІD.• TFІІF and RNAPІІ joins the transcription factors bound at the

promoter.• TFІІE joins the transcription complex and finally TFІІH and TFІІJ join

the promoter. • Formation of closed promoter (binary) complex.• Closed complex converted into open promoter complex.• Around this stage TFІІE is lost. ATP hydrolysis may lead to the loss of

some more factors from the initiation complex.

• RNAP now begins the transcription at the start point and presumably

the transcription factors are released at this stage.

Elongation mechanism in transcription:

•Once eukaryotic RNAPs have been released from their initiation complex they catalyze RNA chain elongation by the same mechanism as RNAP of prokaryotes.

•Early in the elongation process 5' ends of eukaryotic pre-mRNAs are modified by the addition of 7-Methyl Guanosine caps.

7-MG caps are recognized by protein factors involved in initiation of translation and also protect the growing RNA chains from degradation by nucleases

3 RNA polymerase II ends transcription when it reaches a termination signal.

These signals are not well understood in eukaryotes.

TERMINATION•The mode of termination of transcription is much less understood than that of initiation.•As in the case of bacterial RNA polymerase, discrete termination events occur in the case of RNA polymerase I and III.•In case of RNA polymerase I, termination events occur at a discrete site >1000 bp downstream of the 3’ end of the precursor RNA.

• Termination mechanism of transcription:• 3' ends of RNA transcripts synthesized by RNAPІІ are produced

by endonucleolytic cleavage of primary transcripts.• Actual transcription termination occur at multiple sites located

1000 to 2000 nucleotide downstream from the 3' end of the mature transcripts i.e., transcription proceeds beyond the site that become 3' terminal and distal segment is removed.

• The cleavage event occurs at a site 11 to 30 nucleotides downstream from a conserved sequence AAUAAA and upstream from a G-U rich sequence located near the end of transcription unit.

• After cleavage, the enzyme poly(A) polymerase adds poly(A) tails – tracts of AMP residues about 200 nucleotides long to the 3' end called polyadenylation.

• Poly(A) tails of eukaryotic mRNA enhance their stability and play an important role in their transport from nucleus to cytoplasm.

•

COMPARISON OF THE TRANSCRIPTION IN PROKARYOTES & EUKARYOTES

CHARACTERISTIC PROKARYOTES EUKARYOTES

RNA polymerase(a)Types(b)Molecular weight(c)subunits

One in each species465 KDα2 β β′ ωσ.

Three. Pol I II AND III500KDTwo large subunits + <10 smaller units

Transcriptional factors Not known Many different types

promoter A simpler and relatively smaller sequence.

Larger sequence, has variable modules

enhancer A part of the promoter may act in a manner similar to enhancer

Present in variable distances from the promoter.

Transcriptional initiation The holo enzyme binds tightly to the promoter and initiates transcription.

Trans. Factors first bind to promoter then RNA polymerase associates with them and initiates.

Transcriptional complex(a)Composition Core enzyme + sigma

factorRNA pol + TF

(b) Separation of components

Sigma factor dissociates from the core enzyme after initiation

TF dissociates when transcription is initiated

End product Polycistronic RNA transcripts

Monocystronic RNA transcripts

REVERSE TRANSCRIPTION

Reverse transcriptase creates single-stranded DNA from an RNA template.In virus species with reverse transcriptase lacking DNA-dependent DNA polymerase activity, creation of double-stranded DNA can possibly be done by host-encoded DNA polymerase δ, mistaking the viral DNA-RNA for a primer and synthesizing a double-stranded DNA by similar mechanism as in primer removal, where the newly synthesized DNA displaces the original RNA template.The process of reverse transcription is extremely error-prone and it is during this step that mutations may occur. Such mutations may cause drug resistance.

In eukaryotesSelf-replicating stretches of eukaryotic genomes known as retrotransposons utilize reverse transcriptase to move from one position in the genome to another via an RNA intermediate. They are found abundantly in the genomes of plants and animals. Telomerase is another reverse transcriptase found in many eukaryotes, including humans, which carries its own RNA template; this RNA is used as a template for DNA replication.

In prokaryotesInitial reports of reverse transcriptase in prokaryotes came as far back as 1971 (Beljanski et al., 1971a, 1972). These have since been broadly described as part of bacterial Retron msr RNAs, distinct sequences which code for reverse transcriptase, and are used in the synthesis of msDNA. In order to initiate synthesis of DNA, a primer is needed. In bacteria, the primer is synthesized during replication

GENETICS VERMA AND AGARWAL.GENETICS SNUSTARD AND SIMMONS.WEB.

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