lecture 4: dna transcription 1) what is the central dogma of molecular biology 2) what are the steps...
TRANSCRIPT
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
E:\Lessons\5-4_Transc-Transl-b3\Transc-Transl.swf
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