Lecture 4: DNA transcription

Reference:Essential Cell BiologyEssential Cell Biology by Alberts et al

Central dogma of molecular biology

What is the pathway from DNA to Protein?What is the pathway from DNA to Protein?

Transcription- Transcription- DNA directed RNA DNA directed RNA synthesissynthesis

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

place and level of protein expression

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 (mRNA, tRNA and rRNA)

Where does transcription take place?Where does transcription take place?

A) transcribing a primary RNA transcript

B) modification of this transcript into mRNA (transcriptional processing)

Transcription in eukaryotes

Basic structure of a eukaryotic geneBasic structure of a eukaryotic gene

Regulatory region coding region

A. Initiation RNA polymerase binds to promoter & opens helix

B. Polymerisation De novo synthesis using rNTPs as substrate

Chain elongation in 5’-3’ direction

C. Termination stops at termination signal

What are the key events during What are the key events during transcription?transcription?

Question In what direction are the RNA polymerases moving in this image?

A) Initiation by A) Initiation by RNA polymerase RNA polymerase holoenzymeholoenzyme

Prokaryotes:single multisubunit complex

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

Eukaryotes: 3 types

What are promoters and What are promoters and terminators?terminators?Region on DNA where RNA polymerase binds to

initiate or terminate transcription

Which strand carries the coding sequence?Which strand carries the coding sequence?

Sequence of promoter determines Sequence of promoter determines directiondirection of RNA polymerase action of RNA polymerase action

PromotersPromoters

Prokaryotes Near 5’ end of

operons Pribnow box –

consensus sequence TATAAT

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

forconstitutive structural genes – GGGCGGSelective structural genes – TATA

Enhancers and silencers Sequences associated with a promoter which

enhance/repress the activity of a promoter due to its association with proteins called transcription factors

Mediate most selective gene expression in eukaryotes

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) Terminationspecific 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

B) Transcriptional processing

3 main steps

1) RNA capping,

2) polyadenylation

3) splicing

1) CappingAddition 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) TailingAddition 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 removalof intron sequences

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

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

Transcriptional processing of

ovalbumin gene

Is an mRNA always processed in the same way?Is an mRNA always processed in the same way?-- “alternative splicing”-- “alternative splicing”

Question: Will counting the number of promoters yield the number of “genes” in the human genome?

Differential splicing in specific tissues

How do mRNAs get to the cytosol?How do mRNAs get to the cytosol?

Figure 15-8Why do eukaryotes have DNA within a membrane bound compartment and prokaryotes do not?Could eukaryotes function without it?

During "RNA processing"

A. all of the exons are removed B. the RNA molecule is made from a DNA template.C. introns are removed from the RNA and the exons are spliced together.

D. the RNA molecule is translated into a protein molecule.

Since the two strands of the DNA molecule are complementary

A. The RNA polymerase can bind to either strand.

B. Only one strand actually carries the genetic code for a particular gene.

C. Each gene possesses an exact replica that can be used should a mutation occur.

D. A gene transcribed in the 5’ to 3’ direction on one strand can be transcribed in the 3’ to 5’ direction on the other strand.

The presence of a poly-A tail on a RNA molecule indicates that

A. there are exons present that must be removed.

B. this RNA molecule does not contain introns.

C. the transcript should be immediately degraded.

D. this is a rRNA molecule.E. None of the above answers is correct

. “Alternative splicing” refers to

A. the use of introns as exons, or vice versa, during RNA processingB. splicing out of damaged DNA by DNA repair enzymes.C. joining of RNA from two different genes to form a new mRNA.D. the use of alternative reading frames when translating an mRNA.E. a new dance for people with alternative life styles.