BB10006-Cell biology Dr. MV Hejmadi ()

Lecture 4: DNA transcription

1) What is the central dogma of molecular biology

2) What are the steps involved in transcribing a eukaryotic 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

What is the biological significance of transcription?

·  Allows selective expression of genes

·  Regulation of transcription controls time, place and level of protein expression


E.g. Regulation of cellular response to hypoxia (during cerebral ischaemia/stroke)

What is 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.

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.

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.

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

Transcription in eukaryotes uses 2 steps

Step 1: transcribing a primary RNA transcript

Step 2: modification of this transcript into mRNA

Step 1 – Transcribing a primary RNA transcript - An overview of the 3 steps involved

A) Initiation by RNA polymerase holoenzyme (an agglomeration of many different factors that together direct the synthesis of mRNA on a DNA template and which has a natural affinity for DNA) binding to specific DNA sequences called promoters that drive transcription (region where RNA polymerase binds to initiate transcription). The sequence of promoter determines direction of RNA polymerase action. Rate of gene transcription depends on rate of formation of stable initiation complexes

B) 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

C) Termination: Specific termination sequence e.g E.coli needs 4-10A followed by a palindromic GC rich region as well as additional termination proteins such as

e.g. Rho factor in E.coli

a)  Step 2:

Key features of post transcriptional processing are

·  Control of gene expression following transcription but before translation

·  Conversion of primary transcript (hnRNA) into mature mRNA

·  Occurs primarily in eukaryotes

·  Localised in nucleus

Modification (post-translational processing) involves 3 main steps

Ø  RNA capping,

Ø  polyadenylation

Ø  Splicing

1) Capping

Addition of 7 methylguanosine at 5’ end

Mediated by guanylyltransferase

Probably protects against degradation

Serves as recognition site for ribosomes

Transports mRNA 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

RNA polymerase

Prokaryotes have a single multisubunit RNA polymerase complex whereas in eukaryotes 3 types exist

Gene expression efficiency- How is transcription controlled? E.g. When to transcribe gene? How many copies to be transcribed?

DNA binding proteins usually regulate transcriptional activity. These are proteins that recognise & bind to specific sequences on DNA. Recognition is 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