Download Lecture 4: DNA transcription

BB10006-Cell biology
Dr. MV Hejmadi ([email protected])
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)
Basic structure of a gene
Regulatory region
coding region
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
BB10006-Cell biology
Dr. MV Hejmadi ([email protected])
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
 RNA capping,
 polyadenylation
 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
BB10006-Cell biology
Dr. MV Hejmadi ([email protected])
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