The discovery of the role of RNA RNA structure, synthesis and function

Transcription

1 Central Dogma The discovery of the role of RNA RNA structure, synthesis and function! Fundamental observations in genetics!! Genes are located in nuclei (in eukaryotes)!! Polypeptides are synthesised in the cytoplasm!! Genes are made of DNA - a gene is a stretch of DNA!! Each polypeptide is specified by one gene - a polypeptide is a sequence of amino acids! Cytoplasm Nucleus One gene one polypeptide Two information puzzles 1!!!!Transfer of information:!how can genes made of DNA and located in the nucleus direct the! synthesis of proteins in the cytoplasm?! 2!!!!Translation of information:!how can a! sequence of bases (a gene) code for a sequence of amino acids (a polypeptide)?!!!there is no obvious structural relationship! between a base and an amino acid!! How can DNA in the Nucleus Specify the Production of Proteins in the Cytoplams? Is there an intermediate? The idea that RNA acts as an intermediate was suggested by the following findings in eukaryotic cells: 1.! DNA is mostly associated with chromosomes in the nucleus while ribosomes that generate proteins are in the cytoplasm. 2.! RNA is synthesised in the nucleus. 3.! RNA migrates to the cytoplasm where proteins are synthesised. 4.! The amount of RNA is generally proportional to the amount of protein in a cell.

2 One gene: one polypeptide! Opposite DNA strands can serve as template for RNA DNA makes RNA makes protein RNA is a polymer RNA is a polymer Ribose (a sugar) Bases - 4 kinds Usually a single strand Purines Adenine Guanine Pyrimidines Cytosine Uracil Phosphate May have intramolecular double-stranded regions Uracil (U) like T is complementary to A

3 ! The four ribonucleotides found in RNA Figure 8-2 Similarities and Differences Between DNA and RNA RNA! DNA!! Similar strand structure"! Can define a 5# and 3# end"! 2# hydroxyl in RNA: causes stability differences)"! Uracil in RNA takes the place of Thymine in in DNA" 2 OH 2 H Nucleosides and nucleotides RNA is a polymer Sugar + Base = Nucleoside Sugar + Base + Phosphate = Nucleotide AMP - adenosine monophosphate ADP - adenosine diphosphate ATP - adenosine triphosphate Monomers Nucleoside monophosphates AMP GMP CMP UMP Sugar phosphate backbone RNA is a polymer Monomer Monomers Nucleoside monophosphates Monomers Nucleoside monophosphates AMP GMP CMP UMP AMP GMP CMP UMP A G C U

4 Sugar Base RNA involved in protein synthesis Monomers Phosphate 1) Ribosomal RNA (rrna)! 18S, 28S and 5S rrna! structural and functional components of ribosomes! highly abundant and stable Nucleoside monophosphates AMP A GMP G CMP C UMP U 2) Messenger RNA (mrna)! typically about bases long! encodes protein! multiple types, usually not abundant, unstable 3) Transfer RNA (trna)! very small - less than 100 bases long! key role in translation! abundant and stable Newly discovered Classes of RNA Main focus of this lecture 1)! Ribozymes RNA molecules which have enzymatic activity 2)! MicroRNAs RNAs which regulate gene expression 3)! 7S RNA Participates in transport of proteins! Synthesis of messenger RNA in prokaryotes!! mrna carries the DNA code for a polypeptide! One gene: one polypeptide The Prokaryotic Transcription Apparatus Need to understand the following:!! Differences between DNA and RNA.!! Differences between DNA and RNA polymerases.! Messenger RNA! E. coli RNA polymerase and its function.!! What sequences make up a prokaryotic promoter.!! The steps of transcription initiation and elongation.! DNA makes mrna makes protein:! The two different mechanisms of transcription termination.!

5 Evidence for the Existence of mrna Base pairing explains DNA replication and RNA synthesis. Sequence in new DNA or RNA is complementary to the template. In 1956 & 1958, Volkin and colleagues undertook studies on bacteriophage infections in E. coli. 1.! 32 P-labelling of newly synthesised RNA showed it closely resembled the base composition of phage DNA. 2.! Newly synthesised RNA is unstable. 3.! Phage RNA synthesis precedes protein synthesis. In 1961 Spiegelman and colleagues demonstrated that the newly synthesised RNA was complementary to the phage DNA and not the bacterial DNA. In the same year, Brenner, Jacob and Meselson demonstrated that the newly synthesised phage RNA associates with bacterial ribosomes and the phage proteins are synthesised on these ribosomes. Synthesis of RNA implies existence of an enzyme 1959 Samuel Weiss and others isolated an enzyme that could synthesise RNA. Called RNA Polymerase. To function RNAP requires: Double-stranded DNA as a template 4 NTPs (ATP, CTP, GTP, UTP). Unlike DNA polymerase, RNA polymerase does NOT require a primer to initiate synthesis. Uses one strand of double stranded DNA as a template. Synthesis of RNA Requirements for RNA synthesis DNA ATP + GTP + CTP + UTP Mg + RNA polymerase Different Types of RNA Polymerase RNA Synthesis is in the 5 to 3 Direction RNA strand DNA strand Subsequent hydrolysis of PPi drives the reaction forward. OH Similar to DNA synthesis ONE STRAND IS COPIED RNA has polarity (5 phosphate to 3 hydroxyl) OH

6 ! Many RNAs can be simultaneously transcribed from a gene Promoter Terminator 5 3 Some nomenclature conventions Prokaryotic Transcription (antisense strand) (noncoding strand) RNAP! (sense strand) (coding strand) RNA synthesis starts at special sequences: promoters RNA synthesis stops at special sequences: terminators Nomenclature! Transcription Initiation Site! 5"! 3"! Upstream " -5! -4! -3! -2! -1! Template strand! Downstream " +1! +2!+3!+4!+5!+6! 3"! 5"! Direction of transcription" There is no zero!

7 What does a promoter look like?! Purify a short DNA molecule! Imagine this DNA has a promoter! Label double stranded DNA molecules with 32P at on one strand on one end! Add RNAP to a sample (+)! RNAP which is big (500,000 daltons) is expected to bind to the promoter! Other sample is control - no RNAP added (-)! Add DNAase for a short time - to cut on average once per strand - it cuts at random at phosphodiester bonds in the backbone of the DNA Radioactive phosphorous on the top strand of DNA Radioactive phosphorous on the top strand of DNA RNAP! P *! -35! RNAP! -35! -10! RNAP! -35! +1! What does a promoter look like? +1! RNAP footprint at promoter P *! DNAase added -10! Start site of transcription is +1 RNAP footprint at promoter P *! RNAP footprint at promoter -10! +1! DNAase cuts backbone of DNA - where it is free Mapping promoters! Note result! Note the ladder of DNA fragments! Each 1 base shorter than one above! There is a gap over several bands! This shows that RNAP protected certain bonds from cleavage by DNAase.

8 RNAP binds a region of DNA from -40 to +20" Here are some real results The sequence of the non-template strand is shown" Compare the two lanes (+) RNAP added (-) no RNAP See the two gaps in the (+) ladder Start Numbers are the distances in bases from the start point of transcription Conclude that RNAP covers two regions upstream of the start site The C lane shows the start site of transcription (+1) -10 region! TTGACA bp... TATAAT" -35 spacer -10 " Important Promoter Features (tested by mutations)! The closer the match to the consensus the stronger the promoter (-10 and -35 boxes)! The absolute sequence of the spacer region (between the -10 and -35 boxes) is not important! The length of the spacer sequence IS important: TTGACA - spacer (16 to 19 base pairs) - TATAAT! Spacers that are longer or shorter than the consensus length make weak promoters Properties of Promoters! Promoters typically consist of a 40 bp region on the 5'-side of the transcription start site! Two consensus sequence elements:! The "-35 region", with consensus TTGACA! The Pribnow box near -10, with consensus TATAAT - this region is ideal for unwinding. RNA polymerase has many functions Structure of RNAP in Prokaryotes! Scans DNA and identifies promoters! Binds to promoters! Initiates transcription! Elongates the RNA chain! Terminates transcription! Is responsive to regulatory proteins (activators and repressors) As might be expected, RNAP is a multisubunit enzyme! E.coli RNAP is a 465 kd complex! Sub-units: 2!, 1 ", 1 "', 1 # (holoenzyme).! Core enzyme is 2!, 1 ", 1 " (can transcribe but it cannot find promoters).! # recognizes promoter sequences on DNA; "' binds DNA; " binds NTPs and interacts with #.!! subunits appear to be essential for assembly and for activation of enzyme by regulatory proteins.

9 !%$! $! 2 $! 2 " $! 2 "" = core enzyme! "$ "! vegetative! (principal #)! Assembly of RNAP: sigma factors The assembly pathway of the core enzyme!!%%$ CORE ENZYME! Sequence-independent,! nonspecific transcription! initiation! +! # 70 $ # 32 $ # 60 $ SIGMA SUBUNIT! heat shock! (for emergencies)! nitrogen starvation! (for emergencies)! interchangeable,! promoter recognition!!%$ In the Holoenzyme:!!%%$ "$ "! # 70 $ RNAP HOLOENZYME -# 70! Promoter-specific! transcription initiation!! "' binds DNA!! " binds NTPs!! " and " ' together make up the active site!!! subunits appear to be essential for assembly and for activation of enzyme by regulatory proteins. They also bind DNA.!! # recognizes promoter sequences on DNA! Binding of polymerase to template DNA! Polymerase binds nonspecifically to DNA with low affinity and migrates, looking for promoter.! Sigma subunit recognizes promoter sequence.! RNA polymerase holoenzyme and promoter form "closed promoter complex" (DNA not unwound).! Polymerase unwinds about 12 base pairs to form "open promoter complex. Finding and binding the promoter"! Transcription initiation in prokaryotes Closed complex formation" RNAP bound -40 to +20" Open complex formation" RNAP unwinds from -10 to +2" Binding of 1st NTP" Requires high purine [NTP]" Addition of next NTPs" Requires lower purine [NTPs]" Dissociation of sigma" After RNA chain is 6-10 NTPs long"

10 ! Elongation and termination of transcription Chain Elongation Core polymerase - no sigma! Polymerase is pretty accurate - only about 1 error in 10,000 bases (not as accurate as DNAP III)! This error rate is acceptable - many transcripts are made from each gene! Elongation rate is bases per second - slower in G/C-rich regions and faster elsewhere! Topoisomerases precede and follow polymerase to relieve supercoiling Chain Termination Two mechanisms Rho-Dependent Transcription Termination" (depends on a protein AND a DNA sequence)! G/C -rich site" 1) Rho - dependent!rho, a termination factor, is an ATP-dependent helicase!it moves along the RNA transcript, finds the "bubble", unwinds it and releases the RNA chain. RNAP slows down" Rho helicase catches up" Elongating complex is disrupted" Chain Termination 2) Rho-independent termination Rho-Independent Transcription Termination! (depends on DNA sequence - NOT a protein factor)! - termination sites in DNA!inverted repeat, rich in G:C, which forms a stem-loop in RNA transcript!6-8 A s in DNA coding for U s in transcript Stem-loop structure"

11 Rho-independent transcription termination!! RNAP pauses when it reaches a termination site."! The pause may give the hairpin structure time to fold"! The fold disrupts important interactions between the RNAP and its RNA product"! The U-rich RNA can dissociate from the template"! The complex is now disrupted and elongation is terminated" Major Differences between prokaryotic and eukaryotic transcription Differences between prokaryotic and eukaryotic transcription Nuclear membrane DNA is complexed with histones in nucleosomes Promoters have many control sites for transcriptional factors RNAP has many co-factors Precursor to mrna has INTRONS and EXONS The INTRONS are spliced out in the nucleus mrna has a 5 CAP structure and a 3 polya tail mrna transported from nucleus to cytoplasm! Prokaryotic and eukaryotic transcription and translation compared! Transcription initiation in eukaryotes

12 ! Transcription initiation in eukaryotes! Transcription initiation in eukaryotes! Cotranscriptional processing of RNA! Cotranscriptional processing of RNA! Cotranscriptional processing of RNA! Cotranscriptional processing of RNA

13 ! Alternative splicing: alpha tropomyosin Transcription Regulation in Prokaryotes! Why is it necessary?! Bacterial environment changes rapidly.! Survival depends on ability to adapt.! Bacteria must make the proteins required to survive in that environment.! Protein synthesis is costly (energetically).! Therefore, want to make enzymes when required. Proteins Ways to Regulate Transcription 1. Alternate sigma factors: controls selective transcription of entire sets of genes vegetative (principal) s 70 TTGACA +1 (16-19 bp) TATAAT (5-9 bp) A heat shock s 32 CNCTTGA +1 (13-15 bp) CCCATNT (5-9 bp) A nitrogen starvation s 60 CTGGNA +1 (6 bp) TTGCA (5-9 bp) A Ways to Regulate Transcription! 2. Positive Regulation (activation): a positive regulatory! factor (activator) improves the ability of RNAP to! bind to and initiate transcription at a weak promoter.! Ways to Regulate Transcription! 3. Negative Regulation (repression): a negative regulatory! factor (repressor) blocks the ability of RNAP to! bind to and initiate transcription at a strong promoter.! Activator! RNAP! RNAP! -35! -10! +1! Repressor! Activator binding site! -35! -10! +1! EXAMPLE: CAP EXAMPLE: lac REPRESSOR Operator!

14 Protein Synthesis is Regulated Transcriptionally Protein Synthesis is Regulated Transcriptionally! Genes that encode proteins with related functions are grouped in bacteria into transcriptional units called operons!! This ensures that genes for enzymes in the same metabolic pathway are all made at the same time" Operons have three functional parts! 1)! structural genes: these encode proteins (usually with related functions)" 2) Promoter" 3) Regulatory sequences that interact with regulatory proteins" 4) An operon may be associated with regulatory genes which encode proteins regulating expression of that operon" Architecture of a typical operon! promoter" Structural genes" RNA transcript covers all genes in the operon" = polycistronic RNA " Operator (regulatory sequence that binds a repressor protein)" By regulating a single promoter you can co-ordinate " the expression of three genes (in this example)"