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

RNA5’

5’

5’3’

3’

3’DNA

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 1st 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 13

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 ProcessingAdditional modification in RNA after transcription1)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 codeImportant 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-tRNAfmet - for initiation AUG

tRNA met – internal AUG

• Initiation in bacteria require:

70S rib, mRNA, fmet-tRNAfmet

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

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Initiation1) 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-tRnafmet can bind

• fMet-tRnafmet 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-tRnafmet

• 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 2nd 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 2nd 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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