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 gene Regulatory region coding region

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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….. 3) Ribosomal RNA (rRNA) 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

Where does transcription take place?

Transcription in eukaryotes Step 1: transcribing a primary RNA transcript Step 2: modification of this transcript into mRNA

Step 1 - overview Initiation Polymerisation 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

PROMOTERS Prokaryotes Fig 29-10: Voet and Voet Near 5’ end of operons Pribnow box – consensus sequence TATAAT Fig 29-10: Voet and Voet

PROMOTERS Eukaryotes Near 5’ end of genes Recognised by RNA pol II Consensus promoter sequence for constitutive structural genes – GGGCGG Selective 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 sequence e.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 Post transcriptional processing 3 main steps RNA capping, polyadenylation splicing

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 Prokaryotes single 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 Synthesises most rRNA precursors Synthesises mRNA precursors Synthesises 5S rRNA, tRNA, snRNAs

(RNA)n + rNTP = (RNA)n+1 + Ppi 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 include Transcription factors Proteins that recognise & bind to specific DNA sequences Recognition determined by specific structural motifs e.g. helix – loop –helix, zinc finger, leucine zipper Examples include Transcription factors general transcription factors Upstream transcription factors Inducible transcription factors Activators Repressors (silencers)

How does transcriptional control differ in pro and eukaryotes? Prokaryotes Genes are usually switched ‘on’ by default Repressor proteins needed to ‘stop’ transcription Eukaryotes Genes 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