RNA: Structure & Synthesis

By

Amr S. Moustafa, M.D.; Ph.D.

Objectives

The differences between DNA and RNA

The structure and functions of RNAs

RNA synthesis (Transcription)

Post-transcriptional events (modifications)

RNA & DNA: Similarity

Both RNA & DNA:Unbranched polymers

Nucleoside monophosphate

Phosphodiester bonds

RNA & DNA: Differences

RNASingle-Strand (mostly)Cytoplasm (mainly)AGCU (exception)Modified basesRiboseProtein BiosynthesisPost-transcriptional events

DNADoubleNucleusd AGCT

DeoxyriboseStorage &transferDNA Repair

Gene Expression - 1

Gene Expression - 2

From Nucleusto

Cytoplasm

DNA(Gene)

(1) mRNA

(2) Protein

Information Pathway

RNA: TypesMajor types:

Ribosomal RNA (rRNA) 80%

Transfer RNA (tRNA) 15%

Messenger RNA (mRNA) 5%

Others:Small nuclear RNA (snRNA)

Nucleoprotein complexes

of ribosomes

Svedberg Unit:

The rRNA

Related toMolecular weight

& Shape

The tRNASmallest RNA 4S (74 – 95)

At least 20 species

Unusual bases

Secondary structure Intra-chain base pairingAdaptor molecule Carries its sp. a.a. to site of protein biosynthesis

The mRNASize:Heterogeneous (500 – 6000)

Primary (precursor): hnRNA

Post-transcriptionalProcessing of

Euokaryotic mRNA

Carries genetic information from nucleus to cytoplasm(Template of protein synthesis)

RNA Synthesis (Transcription)

Transcription: the copying process which uses one of the two DNA strands (template strand) to form RNAOther DNA strand: coding strand Similar to RNA strand (with exception)

Transcription is highly selective: Some DNA regions form many, other form few or no transcripts

Transcription - 2

The selectivity is due to signals in the DNA nucleotide sequence

The signals convey a message toRNA polymerase to define: Where polymerase should start?

How often? (frequency)

Where to stop? (termination)

Transcription – 3Prokaryotic RNA

polymerase

Transcription – 4Prokaryotic RNA

polymeraseOne species for all RNAs (Except primase for RNA primer)

Elongation: 5’– 3’ polymerizing activity

Initiation: Recognition of promoter sequence

Termination: ρ(rho)-independent

No exonuclease or proofread activity

Transcription:

Transcription – 5

Complementary and

antiparallelDNA and RNA

Transcription – 6Prokaryotic Promoter

Promoter is DNA sequence recognized by (σ) Sigma factor of RNA polymerase holoenzyme where transcription should start

Consensus, highly conserved sequences-10 Pribnow box (TATAAT)-35 Sequence (TTGACA)

(as read 5’ –3’ on nontemplate ‘coding’ strand)

Mutant promoter affects transcription of its gene

Transcription – 7Prokaryotic Promoter

Consensus, highly conserved sequences

Transcription – 8Transcription Unit

Transcription Unit: DNA region from promoter to termination

The product: primary transcript

Transcription – 9Initiation

Binding of RNA polymerase holoenzyme to promoterLocal unwindingSupercoils (Topoisomerases I & II)

Denovo synthesis: No primer required

Substrate: Ribonucleoside triphosphate

Usually begins with a purine

No proofreading

Once promoter is recognized, Sigma (σ) factor is released

Transcription – 10Elongation

Direction: 5’– 3’, antiparallel

Complementarity: as DNA, but for A there is U release of pyrophosphate

Relaxation of supercoils:

Topoisomerases I and II

Transcription – 11Termination

A-The rho (ρ)-dependent termination:The ρ-factor binds to C-rich near 3’-end of RNA

It has ATP-dependent DNA-RNA helicase activity

Hydrolyzes ATP (Energy-dependent)Unwind 3’-end of the transcript from template

Movement of (ρ) protein along RNA/DNA hybrid 5’-3’

Displacement of DNA template at termination site, releasing of RNA transcript

Transcription – 12Termination (Cont’d)B-The rho-independent termination:

Transcription – 13Inhibitors

(Antibiotics)Rifampin (anti-tuberculous): binds to the β-subunit interferes with formation of first phosphodiester bond inhibit RNA synthesis and bacterial growth

Dactinomycin (Actinomycin D): binds to minor groove of DNA template interferes with movement of RNA polymerase inhibits prokaryotic transcription (antibiotic) inhibits euokaryotic replication (anticancer)

Transcription – 14

Rifampin inhibits RNA polymeraseand transcription