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Lecture 4: DNA transcription

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1 Lecture 4: DNA transcription
Reference: Essential Cell Biology by Alberts et al

2 Central dogma of molecular biology

3 What is the pathway from DNA to Protein?

4 Transcription- DNA directed RNA synthesis
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) What is the biological significance? Allows selective expression of genes Regulation of transcription controls time, place and level of protein expression

5 Where does transcription take place?

6 Transcription in eukaryotes
A) transcribing a primary RNA transcript B) modification of this transcript into mRNA (transcriptional processing)

7 Basic structure of a eukaryotic gene
Regulatory region coding region

8 What are the key events during transcription?
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 Question In what direction are the RNA polymerases moving in this image?

9 A) Initiation by RNA polymerase holoenzyme
Prokaryotes: single multisubunit complex Eukaryotes: 3 types 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

10 What are promoters and terminators?
Region on DNA where RNA polymerase binds to initiate or terminate transcription

11 Sequence of promoter determines direction of RNA polymerase action
Which strand carries the coding sequence?

12 Promoters Eukaryotes Prokaryotes Near 5’ end of genes
Recognised by RNA pol II Consensus promoter sequence for constitutive structural genes – GGGCGG Selective structural genes – TATA Prokaryotes Near 5’ end of operons Pribnow box – consensus sequence TATAAT

13 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

14 B) Polymerisation RNA polymerase binds to promoter & opens helix
RNA polymerase catalyses addition of rNTPs in the 5’-3’ direction RNA polymerase generates hnRNAs (~ nt long) & all other RNAs Stops at termination signal

15 C) 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

16 B) Transcriptional processing
3 main steps RNA capping, polyadenylation splicing

17 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

18 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

19 3) Splicing Highly precise removal of 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

20 Transcriptional processing of ovalbumin gene

21 Is an mRNA always processed in the same way? -- “alternative splicing”
Differential splicing in specific tissues Question: Will counting the number of promoters yield the number of “genes” in the human genome?

22 How do mRNAs get to the cytosol?
Figure 15-8 Why do eukaryotes have DNA within a membrane bound compartment and prokaryotes do not? Could eukaryotes function without it?

23 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.

24 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.

25 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

26 . “Alternative splicing” refers to
A. the use of introns as exons, or vice versa, during RNA processing B. 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.

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