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 genesRegulation of transcription controls time, place and level of protein expression
6 Transcription in eukaryotes A) transcribing a primary RNA transcriptB) modification of this transcript into mRNA (transcriptional processing)
7 Basic structure of a eukaryotic gene Regulatory regioncoding region
8 What are the key events during transcription? Initiation RNA polymerase binds to promoter & opens helixB. Polymerisation De novo synthesis using rNTPs as substrateChain elongation in 5’-3’ directionC. Termination stops at termination signalQuestionIn what direction are the RNA polymerases moving in this image?
9 A) Initiation by RNA polymerase holoenzyme Prokaryotes:single multisubunit complexEukaryotes: 3 typesRNA pol IRNA pol IIRNA pol IIILocated in nucleoliLocated in nucleoplasmSynthesises most rRNA precursorsSynthesises mRNA precursorsSynthesises 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 IIConsensus promoter sequence forconstitutive structural genes – GGGCGGSelective structural genes – TATAProkaryotesNear 5’ end of operonsPribnow 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 factorsMediate 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’ directionRNA polymerase generates hnRNAs (~ nt long) & all other RNAsStops at termination signal
15 C) Termination specific termination sequence e.g E.coli needs 4-10A followed by a palindromic GC rich regionAdditional termination proteinse.g. Rho factor in E.coli
16 B) Transcriptional processing 3 main stepsRNA capping,polyadenylationsplicing
17 1) Capping Addition of 7 methylguanosine at 5’ end Mediated by guanylyltransferaseProbably protects against degradationServes as recognition site for ribosomesTransports hnRNA from nucleus to cytoplasm
18 2) Tailing Addition of poly(A) residues at 3’ end Transcript cleaved 15-20nt past AAUAAAPoly(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
21 Is an mRNA always processed in the same way? -- “alternative splicing” Differential splicing in specific tissuesQuestion:Will counting the number of promoters yield the number of “genes” in the human genome?
22 How do mRNAs get to the cytosol? Figure 15-8Why 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 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.