Review

• DNA is the storage molecule for genetic information

• DNA replication – process of copying genetic information to pass on to daughter cells • DNA must be replicated with high fidelity (no mistakes)

• DNA must allow access to genes for expression, needs to be selective as only certain genes are active in certain cells

• Central dogma of gene expression

DNA RNA Protein

Review

• Structure of DNA – polymer of deoxyribonucleic monophosphates • Long, unbranched chain with polarity

• Linked together by a 3’ – 5’ phosphodiester bond

• DNA is a double helix • Twisted ladder

• Hydrophilic (negatively charged) phosphodiester backbone is solvent exposed

• Hydrophobic bases form rungs of ladder inside the helix

Review

• Base-pairing • A-T, G-C, purines = pyrimidines

• H-bonds between bases, 2 for A-T and 3 for G-C

• Bases are stacked in the helix and hydrophobic interactions between the stacked bases further stabilize the helix

• Heat and extreme pH can “melt” the helix by disrupting the H-bonds between bases • Higher G-C content more stable

Review

• DNA synthesis • Strand separation – DnaA, helicase and ssDNA binding

proteins

• Replication fork formation – unwinding/opening of strands allow DNA synthesis to occur starting at ori in opposite directions

• Supercoiling prevents DNA synthesis • Topoisomerases nick DNA to relieve coiling then re-ligate

• DNA Replication • Leading strand – copied in direction of advancing fork –

continuous

• Lagging strand – copied away from the fork - discontinous

Review

• RNA primers • Polymerases cannot initiate synthesis from single

stranded template

• RNA primer that is complementary to template is inserted to create a free 3’OH on ds template

• Synthesized by primase

• Removed after synthesis of DNA complete by DNA polymerase I

• Chain elongation • Catalyzed by DNA polymerase III

• Addition of dNTPs in sequence determined by template

• DNA pol III reads 3’-5’ and creates new 5’-3’ strand

Review

• Proofreading • DNA pol III proofreads its work

• Excises incorrect nucleotides as they are put in, and replaces them with correct nucleotide

• Packaging of DNA in Eukaryotic cells • DNA bound to histones to form beads on a string

• Further condensed to 30 nm fibre

• Fibre forms loops that are anchored to a nuclear scaffold protein

• Packaged into chromosomes

RNA Structure and Synthesis – Lecture Objectives 1. Understand the role of RNA in storage and

expression of genetic information

2. Understand the structure of RNA 1. mRNA, tRNA and rRNA

3. Understand the synthesis of RNA 1. Prokaryotic vs Eukaryotic

RNA

• DNA is the blueprint of an organism

• RNA is the “working copies” of the DNA • Express the master plan

• Transcription: the process of copy DNA template to RNA

• Transcription is highly selective • Only specific regions of DNA are expressed

• Selectivity results in the differentiation of cells into different tissues

RNA

• 3 types of RNA 1. Messenger RNA (mRNA) – carries

genetic information from nuclear DNA to cytosol where acts as a template for protein synthesis.

2. Ribosomal RNA (rRNA) – structural part of ribosomes where protein synthesis takes place.

3. Transfer RNA (tRNA) – an adaptor molecule that carries specific amino acids to the ribosomes for protein synthesis.

• rRNA and tRNA are not translated into protein

RNA Structure

• RNAs are unbranched polymers of ribonucleotides joined together by phosphodiester bonds

• RNA differs from DNA: 1. RNA is smaller than DNA

2. Contains ribose instead of deoxyribose

3. Contains uracil instead of thymine

4. RNA is single stranded and capable of folding into complex structures

rRNA

• Ribosomal RNA is associated with proteins to form the components of the ribosomes • Ribosomes are responsible for protein synthesis

• 3 rRNA species in prokaryotics • 23S, 16S and 5S

• 4 rRNA species in eukaryotes • 28S, 18S, 5.8S and 5S

• S refers to Svedberg unit • Related to molecular weight and shape of

compound

• rRNA constitute approx. 80% of the total RNA in a cell

tRNA

• Transfer RNA are the smallest of the 3 major types of RNA • 74- 95 nucleotides in length

• At least one specific tRNA for each of the 20 amino acids

• Constitute approx. 15 % of the total RNA in the cell

• tRNAs contain unusual bases and have extensive intrachain base-pairing • Results in characteristic secondary and tertiary

structure

• Each tRNA cariries a specific amino acid on its 3’ end to the ribosomes • Recognizes 3 base genetic code within the mRNA

which are specific for the addition of a particular amino acid to the growing protein chain

mRNA

• Messenger RNA transports genetic information from DNA in nucleus to the cytoplasm • Template for protein synthesis

• Constitutes approx. 5% of total RNA • Most heterogeneous of RNAs in terms of

size and sequence • 500-6000 nucleotides

• Eukaryotic mRNAs (not prokaryotic) have a long sequence of adenine residues at the 3’ end and a 7-methyl guanosine ‘cap’ attached by triphosphate bond to 5’ end • Poly-A tail and 5’ cap

Transcription of Prokaryotic Genes

• Structure of RNA polymerase, signals controlling transcription and modification to RNA differ between prokaryotes and eukaryotes

• Prokaryotic RNA polymerase synthesizes all 3 major RNA forms

• RNA polymerase is a multi-subunit enzyme that recognizes the promoter region of DNA • Makes complementary RNA copy of

DNA template strand • Reads 3’-5’ and synthesizes 5’-3’

• Transcription unit extends from promotor to termination region of DNA

Transcription by RNA Polymerase

• Involves core enzyme and numerous auxiliary proteins

1. Core enzyme: 5 of the enzyme’s protein subunits combined are responsible for 5’ to 3’ RNA polymerase activity • Lacks specificity

2. Holoenzyme: Binding of σ-factor enables RNA polymerase to recognize promoter regions on DNA template

Steps in RNA Synthesis

• Transcription in E. coli involves 3 steps: 1) initiation; 2) elongation; 3) termination

1. Initiation: Involves binding of RNA polymerase holoenzyme to promoter region of a gene • Promoter region contains consensus sequences that are

recognized by the σ factor a) -35 sequence (5’TTGACA3’): centred ~35 nucleotides

upstream of transcription start site – initial point of contact of RNA polymerase

b) Pribnow box (5’ TATAAT3’): sits 5’ of the transcription start site is the site of initial melting of DNA allowing transcription bubble to form.

Steps in RNA Synthesis

2. Elongation: RNA polymerase begins to synthesize transcript of DNA template and the σ-factor is released • RNA polymerase uses ribonucleoside triphosphates and

releases pyrophosphate (PPi) as each nucleotide is added

• Binding of RNA polymerase induces unwinding of DNA helix

• Supercoiling relaxed by topoisomerases

Steps in RNA Synthesis

3. Termination: elongation continues until termination signal is reached. • Rho factor may or may not be required for released of

newly synthesized RNA

• Rho Dependent Termination: Requires Rho factor to bind to C-rich region near 3’ end of RNA. • Rho factor migrates behind RNA polymerase in 5’ to 3’

direction until termination signal is reached

• At termination signal Rho factor facilitates dissociation of RNA from the DNA template

Steps in RNA Synthesis

• Rho-Independent termination: most common termination

• Sequence in DNA template generates sequence in RNA that is self complementary • Allows RNA to fold back on itself

forming a GC-rich stem • Known as a hairpin

• Beyond the hairpin RNA has a string of Us at the 3’end • Binding to complimentary A’s of DNA

is weak facilitating separation of RNA

Transcription of Eukaryotic Genes

• More complex than for prokaryotes

• In addition to RNA polymerase binding to a promoter region, several other transcription factors must bind to sites on DNA near the promoter region • Binding of different factors determines which genes will

be transcribed

• Double helix must assume a loose conformation and dissociate from nucleosome core for RNA polymerase and transcription factors to bind their DNA sequences

Nuclear RNA Polymerases of Eukaryotic Cells • 3 distinct classes of RNA polymerases in nucleus

1. RNA polymerase I: synthesizes precursors to large rRNAs of ribosomes

2. RNA polymerase II: synthesizes precursors to mRNAs

3. RNA polymerase III: synthesizes precursors of tRNAs and other small RNAs

Promoters for RNA Polymerase II

1. TATA (Hogness) Box – found ~25 nucleotides upstream of transcription start site

2. Inr (initiator) promotor element

3. DPE (downstream promoter element)

• All serve to bind general transcription factors, which interact with each other to recruit RNA polymerase II

Promoters of RNA Pol II • General transcription factors: Minimal

requirements for recognition of the promoter, recruitment of RNA pol II, and initiation of transcription

• Regulatory elements and transcriptional activators: binding sites of proteins that regulate gene expression such as CAAT and GC boxes

Promoters of RNA Polymerase II

• Enhancers: special DNA sequences that increase the rate of initiation of transcription by RNA pol II

• Can be: • Located upstream or downstream of

transcription start site • Close to or 1000s of base pairs away from

promoter • Occur on either strand of DNA

• Bind to STFs (specific transcription factors)

• DNA loops back so the STFs can interact with other transcription factors and RNA Pol II to enhance transcription

Post-transcriptional Modification of RNA • Primary transcript is the initial, linear, RNA copy of

a transcription unit

• Primary transcripts of both prokaryotic an eukaryotic tRNA and rRNA are post-transcriptionally modified • By cleavage by ribonucleases

• mRNA from eukaryotes, but not prokaryotes is also subject to posttranscriptional modification

rRNA Modification

• Ribosomal RNAs are synthesized from long precursors termed preribosomal RNAs

• Preribosomal RNAs are cleaved by ribonucleases (RNases) to produce mature rRNAs

tRNA Modification • Also synthesized from long precursor molecules

• An intron must be removed from anticodon loop and sequences from the ends must be removed.

• Other posttranscriptional modifications include 1. Addition of –CCA sequence to the 3’ end 2. Modification of bases at specific positions to produce

unusual bases

Eukaryotic mRNA Modifications

• Primary transcript undergo extensive co- and posttranscriptional modification in the nuclease

1. 5’ capping: First processing reaction • Guanylyltransferase adds GTP backwards to the 5’ end

by a triphosphate link

• Methylation occurs in cytoplasm to form 7-methyl-guanosine CAP

• Permits initiation of translation and stabilizes the mRNA

Eukaryotic mRNA Modifications

2. Addition of Poly-A tail: most mRNAs have 20 – 200 adenines attached to 3’ end • Added after transcription by nuclear enzyme,

polyadenylate polymerase • Signal sequence (AAUAAA) near 3’end signals addition of

poly A tail • Stabilizes mRNA and facilitates exit from nucleus

3. Removal of introns: • Introns are RNA sequences that do not code for protein • Removed from primary transcript • Coding sequences (exons) spliced together to form

mature mRNA by spliceosome

Removal of Introns from mRNA

1. Role of Small Nuclear RNAs (snRNAs) • Interact with several proteins to form small

nuclear ribonucleoprotein particles (snRNPs) • Form base pairs with consensus sequences

in introns to facilitate splicing of exons

2. Mechanism of Intron Excision • snRNPs bring adjacent exons into correct

alignment for splicing • 2’OH of adenosine in intron attacks and

forms a phosphodiester bond with 5’ phosphate

• Freed 3’OH of exon 1 bonds to 5’ of exon 2 • Excised intron has loop structure that is

broken down • After removal of all introns, mRNA exit

nucleus through nuclear pores

Effect of Splice Site Mutations

• Mutations at splice site can lead to improper splicing and the production of aberrant proteins

• Approximately 15 % of all genetic diseases are a result of mutations that affect RNA splicing

• I.e. mutation that cause incorrect spicing of β-globin mRNA cause some forms of β-thalassemia • Reduced production of hemoglobin

• Errors in splicing can cause removal of exons or retention of introns