Transcription: RNA Synthesis, Processing & Modification

Transcription:

RNA Synthesis, Processing &

Modification

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Central dogma

DNA → RNA → Protein

Reverse transcription

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Transcription

• The process of making RNA from DNA

• Produces all type of RNA –mRNA, tRNA, rRNA, snRNA, miRNA and siRNA

• Ribont is produced rather than deoxyribont

• U replaces T

• A primer is not needed, but a DNA template is needed

• Only a very small portion of the genome is transcribed or copied into RNA – entire genome must be copied during DNA replication

• RNA chain from 5’ to 3’end

• No proofreading

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DNA

5’ RNA

5’

5’

3’

3’

DNA 3’

Template strand/antisense strandy6

Transcription

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3 Major kinds of RNA

• Messenger RNAs (mRNAs) – encode the a.a seq of one or more polypeptide specified by a gene or set of genes

• Transfer RNAs(Trna) – read the information encoded in the Mrna and transfer the

appropriate aa to a growing polypeptide chain during protein synthesis

• Ribosomal RNAs-constituents of ribosomes- cellular machines that synthesize proteins

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• During replication – the entire chromosome is usually copied

• Transcription is more selective

 Only particular genes or groups of genes are transcribed at any one time – some

portions of the DNA genome are never transcribed

 Specific regulatory sequences mark the beginning and end of DNA segments to be transcribed and designate which strand of duplex DNA to be used as the template

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RNA Polymerase

• Synthesized the transcription

• The most studied- in E.Coli

• 5 different subunits –α2ωββ’σ (holoenzyme)

• α2ωββ’– core enzyme

• σ – recognize specific promoter (a DNA sequence that signals the

start of RNA transcription)

• α2ωββ‘ – make the active site for polymerization

• Only holoenzyme can initiate transcription

• Lack the proof reading active site

– more error ⁷

Stages of transcription

• Formation of transcription complex (of DNA and RNA polymerase)

• Initiation

• Elongation

• Termination

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Initiation-RNA synthesis begins at promoters

• RNA Pol need to bind to specific sequence of DNA to start transcription - forms closed complex

• These sequence – promoter

• Sigma factor recognizes the promoter sequence

• Mutation in promoter affect the efficiency of RNAP binding and transcription initiation

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Characteristics of Promoter sequence

• Pribnow box- sequence contained in the promoter region (5-10 bases to the left –

upstream first four bases to be transcribed to RNA)

• All Pribnow box found in eukaryotes are variant of TATAATG sequences – TATA BOX

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Initiation-RNA synthesis begins at promoters

• RNA Pol attaches to promoter region-forms a close complex, promoter DNA is stably bound but not unwound

• RNA Pol melts the helical

structure (~12-15bp from -10 region to +2 and +3) and

separates the 2 strands of DNA locally – open promoter

complex

• RNA Pol initiates RNA synthesis.

The site at which the 1

nt is

added – start site/point

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Chain elongation

• After the strands have separated, a transcription bubble of about 17 bp moves down the DNA sequence to be transcribed

• RNA Pol catalyzes the formation of the phosphodiester bonds between the incorporated ribont

• About 10 nt is added, sigma s/u

dissociates and is later recycled to bind to another RNA Pol core enzyme

• The DNA helix reclosed after RNA Pol transcribes through it and growing RNA

chain dissociates from DNA

Chain termination

1) Intrinsic termination/rho independent termination

•Controlled by termination sites – specific sequences on the DNA molecule function as the signal for termination of transcription process

•Two inverted repeats spaced by few other based followed by repeats of Adenosine

•Inverted repeats – sequences of bases that are complementary, they can loop back on themselves

•When the RNA is created, the inverted repeats form a hairpin loop and stall the advancement of RNA Pol

•The presence of uracils cause a series of A-U base pairs between the template strand and the RNA, and relatively unstable

•RNA dissociate from the transcription bubble- end of transcription ¹⁴

Chain termination

2) The rho (ρ) factor mechanism

• Rho protein binds to the RNA and chases the RNA Pol.

• When the RNA Pol pause at the termination site,

the rho protein has a chance to catch up the RNA Pol

• Rho proteins reaches the termination site, it facilitate the dissociation of the

transcription machinery by unwinds the DNA-RNA hybrid in the transcription bubble

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RNA Processing

Additional modification in RNA after transcription 1)Splicing

• Usually in eukaryotes

• Primary transcript of Mrna contain of intron (non-coding region and exon (coding region)

• Removal of intron by nucleases and joining of exons by ligases- splicing process

• New exons – cont seq that specifies a functional polypeptide

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2) 5’ Cap

• Usually in eukaryote

• 7-methylguanosine linked to the 5’ terminal residue

• 5’ cap helps protect Mrna from ribonucleases

• Also binds to a specific complex of proteins and participates in binding of Mrna to the ribosome to initiate translation

• Occur very early in

transcription, after the first 20/30 nts are added.

RNA Processing

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2) 3’ Poly A tail

• Usually in eukaryote

• 80-250 A residue is added to the 3’end (Poly A tail)

• helps protect Mrna from ribonucleases

RNA Processing

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RNA degradation

• Conc of any molecule depends on rate of synthesis and rate of degradation

• Synth and degradation of an Mrna is balanced – a change in the process lead to accumulation or depletion

• Degradative pathways ensure mrna do not build up in the cell and direct the synthesis of unnecessary proteins

• Degradation depends on the need of the cell

• If needed very briefly-half life of mrna maybe minutes/seconds

• If needed constantly by the cell-can stable for many cell generation

• Average in vertebrate – 3hours

• Average in bacteria – 1.5min

• Degradation by ribonucleases

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TASK

List down the antibiotics that inhibit the process of transcription and explain the mechanism of its action towards inhibiting the pathogen

invasion

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TRANSLATION:

PROTEIN SYNTHESIS AND GENE EXPRESSION

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Introduction

• Protein are end products of most information pathways

• A normal cell need thousand of

different proteins at any given moment

• They must be synthesized in response to the cell’s current needs, transported to their appropriate cellular locations and degraded when no longer needed

• Protein synthesis is a complex process but still are made at exceedingly high rates

• Polyp of 100 res is synth in E.Coli cell in only 5 sec

• 2 key components in protein synth;

ribosome and Trna

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Dictionary of Genetic Code

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Genetic code

Important features:

• Triplet

• Non overlapping

• Commaless- arranged as continuous structure

• Degenerate – dissimilar components can perform a similar fx: UAU and UAC represent tyr

• Universal code

• There are 64 combinations of 3 bases producing 64 codons

• Codons- triplet of nts that codes for a specific aa

• Special codons: AUG (meth – start codon) (UAA, UAG, UGA – stop codon)

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Ribosomes

• E.coli contain >15000 ribosome

• Bact ribosome: 65%

Rrna and 35% proteins

• Bact rib: 70S (50S+30S)

• Euk rib: 80S (60+40S)

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Transfer RNA (TRNA)

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Translation of Mrna

• A process to synthesize a protein from mRNA

• The amino acid (aa) is added sequentially in a specific number and

sequence, determined by the sequence of codons in the genetic code of the relevant mRNA

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STEPS IN PROTEIN SYNTHESIS

• Activation of amino acid

• Initiation

• Elongation

• termination

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Activation of amino acid

• In cytosol, aminoacyl-Trna synthetases esterify the 20aa to their corresponding tRNA

• Each enzyme specific for one aa

• Formation of aminoacyl t-RNA

• The amino acid need to be activated before they can be incorporated into the peptide chain

• Attachment of the correct aa to the adaptor (fidelity)

Amino acid + ATP Aminoacyl AMP + Ppi (aminoacyl-adenylate complex)

Aminoacyl-AMP+t-RNA Aminoacyl Trna + Amp +Ppi

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Initiation

• Prot synth begin at the amino (NH2) terminal and proceeds to the carboxyl (COO) terminal

• AUG (methionin)- start codon

• 2 types of RNA specific for Meth:

 fmet-tRNA^fmet - for initiation AUG

 tRNA ^{met –} internal AUG

• Initiation in bacteria require:

 70S rib, mRNA, fmet-tRNA^fmet

 3 proteins – initiation factors (IF-1, IF-2 and IF-3)

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Initiation

1) Dissociation of ribosome

• Before initiation process starts, 70S ribosome dissociate into 30S and 50S s/u

• 2 initiation factor, IF-3 AND IF-1 binds to the newly dissociated 30S – To

prevent re-association and allows other translation initiation factors to associate with 30S s/u and prepares it for formation of 70S initiation complex

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Initiation

• The association of ribosome and RNA will form preinitiation complex

• Pre initiation complex is guided for initiation codon AUG by Shine Dalgarno sequence

• Precise positioning is needed for initiation

• Bacterial ribosomes have 3 sites:

 Peptidyl (P) – binds a TRNA that carries a peptide chain

 Aminoacyl (A) – binds incoming aminoacyl TRNA

 E (exit)-carries uncharged TRNA that is about to be released from the ribosome

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• The initiating 5’AUG is positioned in at the P site- the only site fMet- tRna^fmet can bind

• fMet-tRna^fmet is the only aminoacyl Trna that binds first to the P site, as during the elongation stage all incoming aminoacyl-trna binds first to the A and only to the P and E

• IF-1 binds at the A site and prevents Trna binding at this site during initiation

Initiation

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Step 2:

• The complex is joined by both GTP bound IF-2 and fMet-tRna

• The anticodon of this Trna now pairs correctly with the Mrna’s

initiation codon

Initiation

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Step 3:

• This complex binds to 50S ribosomal su, and

simultaneously the GTP bound to IF-2 is hydrolyzed to GDP and Pi and released from complex

• All 3 IF depart from rib at this point

• Completion of these steps produces a functional 70S rib – initiation complex

• Now ready for elongation

Initiation

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Elongation

Require:

• The initiation complex

• Aminoacyl-trnas

• Elongation factors (EF-Tu, EF-Ts and EF-G)

• GTP

• Involve 3 steps and cells use these 3 steps to add aa residue and are repeated as many times as

needed

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Step 1:

• Binding of an incoming Aminoacyl- Trna

• The appropriate incoming aminoacyl- TRNA binds to a complex of GTP

bound EF-TU – producing aminoacyl Trna-EF-TU-GTP complex binds to the A site of the 70S initiation complex

• EF-TU-GTP and EF-TU-GDP complexes exist for few ms b4 they dissociate- time for codon anticodon interaction to be proofread.

• Incorrect aminoacyl-Trnas normally dissociate from A site

Elongation

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Step 2: Peptide bond formation

• The peptidyl transferase catalyzing the formation of

peptide bond by transferring N- formylmethionyl group to the amino group of the second

amino acyl-TRNA in the A site – forming dipeptidyl-TRNA

• The uncharged (deacylated) TRNA fmet remains bound to the P site

Elongation

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Step 3: Translocation

• The ribo moves one codon towards the 3’end of the MRNA

• This movement shifts the anticodon of the dipeptidyl Trna (which still attached to the 2^nd codon from A to P site

• At the same time the deacylated Trna is shifted from P to E site, and Trna is releases into the cytosol

• The third codon of the mrna now lies in the A site and the 2^nd codon in the P site

• Movement of rib along Mrna require EF-G (Translocases) and the energy is provided by hydrolysis of another molecule of GTP

• The rib is now ready for next addition of aa

Elongation

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TASK

• List down the antibiotics that inhibit the process of translation and explain the

mechanism of its action towards inhibiting the pathogen invasion

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