Lecture 4: DNA transcription

1) What is the central dogma of molecular biology

2) What are the steps involved in transcribing a primary RNA transcript?

3) How does eukaryotic post-transcriptional processing convert a primary transcript into messenger RNA?

4) Write notes on promoters, enhancers and transcription factors

Central dogma of molecular biology

Transcription

DNA directed RNA synthesis

What is the biological significance?Allows selective expression of genes Regulation of transcription controls time,

place and level of protein expression

Basic structure of a geneBasic structure of a gene

Regulatory region coding region

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Transc-Transl.htm

Transcription Transcription is the mechanism by which

a template strand of DNA is utilized by

specific RNA polymerases to generate

one of the three different types of RNA.

Types of RNA

1) Messenger RNA (mRNA)This class of RNAs are the genetic coding templates used by the translational machinery to determine the order of amino acids incorporated into an elongating polypeptide in the process of translation.

Types of RNA…..

2) Transfer RNA (tRNA)This class of small RNAs form covalent attachments to individual amino acids and recognize the encoded sequences of the mRNAs to allow correct insertion of amino acids into the elongating polypeptide chain.

Types of RNA…..

This class of RNAs are assembled, together with numerous ribosomal proteins, to form the ribosomes. Ribosomes engage the mRNAs and form a catalytic domain into which the tRNAs enter with their attached amino acids. The proteins of the ribosomes catalyze all of the functions of polypeptide synthesis

3) Ribosomal RNA (rRNA)

Where does transcription take place?

Step 1: transcribing a primary RNA transcript

Step 2: modification of this transcript into mRNA

Transcription in eukaryotes

Step 1 - overview

A.Initiation

B.Polymerisati

on

C.

Termination

A) RNA polymerase binds to promoter & opens helix

B) De novo synthesis using rNTPs as substrate

Chain elongation in 5’-3’ direction

C) stops at

termination signal

A) Initiation: ENZYME

RNA polymerase holoenzyme

an agglomeration of many different factors that together direct the synthesis of mRNA on a DNA template

Has a natural affinity for DNA

Initiation: SIGNAL

specific DNA sequences called promoters

1) Region where RNA polymerase binds to initiate

transcription

2) Sequence of promoter determines direction of

RNA polymerase action

3) Rate of gene transcription depends on rate of

formation of stable initiation complexes

PROMOTERSProkaryotes

Near 5’ end of operons Pribnow box – consensus sequence TATAAT

Fig 29-10: Voet and Voet

PROMOTERSEukaryotes

Near 5’ end of genes Recognised by RNA pol II Consensus promoter sequence for

constitutive structural genes – GGGCGGSelective structural genes – TATA

ENHANCERS

Sequences that are associated with a

promoter

Enhance the activity of a promoter due to

its association with proteins called

transcription factors

Enhancers mediate most selective gene

expression in eukaryotes

Polymerisation RNA polymerase binds to promoter & opens helix RNA polymerase catalyses addition of rNTPs in

the 5’-3’ direction RNA polymerase generates hnRNAs (~70-1000 nt

long) & all other RNAs Stops at termination signal

Termination

specific termination sequencee.g E.coli needs 4-10A followed by a palindromic GC rich region

Additional termination proteins e.g. Rho factor in E.coli

Step 2: Modification

3 main steps

1) RNA capping,

2) polyadenylation

3) splicing

Post transcriptional processing

Post transcriptional processing

Control of gene expression

following transcription but

before translation

Conversion of primary

transcript into mature mRNA

Occurs primarily in eukaryotes

Localised in nucleus

Post transcriptional processing

1) Capping

Addition of 7 methylguanosine at 5’ end

Mediated by guanylyltransferase

Probably protects against degradation

Serves as recognition site for ribosomes

Transports hnRNA from nucleus to cytoplasm

2) Tailing

Addition of poly(A) residues at 3’ end Transcript cleaved 15-20nt past

AAUAAA

Poly(A)polymerase and cleavage & polyadenylation specificity factor (CPSF) attach poly(A) generated from ATP

3) Splicing

Highly precise removal of intron sequences

Performed by spliceosomes (large RNA-protein complex made of small nuclear ribonucleoproteins)

Recognise exon-intron boundaries and splice exons together by transesterification reactions

Cell type-specific splicing

Differential splicing in specific tissues

Regulation of gene expression

Prokaryotes• Mainly at

transcriptional level• Sets of genes

transcribed together (polycistronic)

• E.g. lac operon and trp operon in bacteria

Eukaryotes• Other levels of regulation

inlcude posttranscriptional and posttranslational regulation

• Each gene transcribed independently (monocistronic)

RNA polymerase

Prokaryotessingle multisubunit RNA polymerase complex

RNA polymerase

Eukaryotes - 3 types exist

RNA pol I RNA pol II RNA pol III

Located in nucleoli

Located in nucleoplasm

Located in nucleoplasm

Synthesises most rRNA precursors

Synthesises mRNA precursors

Synthesises 5S rRNA, tRNA, snRNAs

RNA polymerase Enzymes that catalyse the formation of RNA using DNA as a template De novo synthesis using rNTP as substrates 1960 – J Hurwitz & S Weiss

(RNA)n + rNTP = (RNA)n+1 + Ppi

Antibiotics such as Rifampicin / rifamycin B inhibit RNA polymerase activity

Gene expression efficiency

When to transcribe gene?

How many copies to be transcribed?

DNA binding proteins

Examples includeTranscription factors• general transcription factors• Upstream transcription factors• Inducible transcription factorsActivatorsRepressors (silencers)

Proteins that recognise & bind to specific DNA sequences

Recognition determined by specific structural motifs

e.g. helix – loop –helix, zinc finger, leucine zipper

How does transcriptional control differ in pro and

eukaryotes?ProkaryotesGenes are usually switched ‘on’ by default Repressor proteins needed to ‘stop’ transcription

EukaryotesGenes are usually switched ‘off’ by default

Transcriptional activators needed to induce transcription

Regulated by chromatin structure, DNA methylation etc

Lac operon

Fig 29-3/5: Voet and Voet

Fig 8-20: Essential Cell Biology by Alberts et al