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 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)
A) transcribing a primary RNA transcript B) modification of this transcript into mRNA (transcriptional processing) Transcription in eukaryotes
Basic 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 transcription? Question In what direction are the RNA polymerases moving in this image?
A) Initiation by RNA polymerase holoenzyme Prokaryotes: single multisubunit complex RNA pol IRNA pol IIRNA pol III Located in nucleoliLocated in nucleoplasm Synthesises most rRNA precursors Synthesises mRNA precursors Synthesises 5S rRNA, tRNA, snRNAs Eukaryotes: 3 types
What are promoters and terminators? Region on DNA where RNA polymerase binds to initiate or terminate transcription
Which strand carries the coding sequence? Sequence of promoter determines direction of RNA polymerase action
Promoters 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 for constitutive structural genes – GGGCGG Selective 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) 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
B) Transcriptional processing 3 main steps 1)RNA capping, 2)polyadenylation 3)splicing
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 snRNPs and spliceosomes (large RNA- protein complex made of small nuclear ribonucleoproteins) Recognise exon-intron boundaries and splice exons together by transesterification reactions
Is an mRNA always processed in the same way? -- “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? Figure 15-8 Why 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 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.