Nucleic Acids: Cell Overview and Core Topics

Outline

I.Cellular Overview

II.Anatomy of the Nucleic Acids1. Building blocks2. Structure (DNA, RNA)

III.Looking at the Central Dogma1. DNA Replication2. RNA Transcription3. Protein Synthesis

Cellular Overview

DNA and RNA in the Cell

Classes of Nucleic Acids: DNA

DNA is usually found in the nucleus

Small amounts are also found in:• mitochondria of eukaryotes• chloroplasts of plants

Packing of DNA:• 2-3 meters long• histones

genome = complete collection of hereditary information of an organism

Classes of Nucleic Acids: RNA

FOUR TYPES OF RNA

• mRNA - Messenger RNA

• tRNA - Transfer RNA

• rRNA - Ribosomal RNA

• snRNA - Small nuclear RNA

Anatomy of Nucleic Acids

THE BUILDING BLOCKS

Nucleic acids are linear polymers.

Each monomer consists of:

1. a sugar

2. a phosphate

3. a nitrogenous base

Nitrogenous Bases

Nitrogenous Bases

DNA (deoxyribonucleic acid):adenine (A) guanine (G)cytosine (C) thymine (T)

RNA (ribonucleic acid):adenine (A) guanine (G)cytosine (C) uracil (U)

Why ?

Properties of purines and pyrimidines:

1.keto – enol tautomerism2.strong UV absorbance

Pentoses of Nucleic Acids

This difference in structure affects secondary structure and stability.

Which is more stable?

Nucleosideslinkage of a base and a sugar.

Nucleotides- nucleoside + phosphate

- monomers of nucleic acids - NA are formed by 3’-to-5’ phosphodiester linkages

Shorthand notation:

- sequence is read from 5’ to 3’- corresponds to the N to C terminal of

proteins

Nucleic Acids: Structure

DNA

Primary Structure

• nucleotide sequences

DNA Double Helix

• Maurice Wilkins and Rosalind Franklin

• James Watson and Francis Crick Features:

• two helical polynucleotides coiled around an axis

• chains run in opposite directions• sugar-phosphate backbone on

the outside, bases on the inside

• bases nearly perpendicular to the axis

• repeats every 34 Å• 10 bases per turn of the helix• diameter of the helix is 20 Å

Secondary Structure

Double helix stabilized by hydrogen bonds.

Which is more stable?

Axial view of DNA

A and B forms are both right-handed double helix.

A-DNA has different characteristics from the more common B-DNA.

• left-handed• backbone phosphates zigzag

Z-DNA

Comparison Between A, B, and Z DNA: A-DNA: right-handed, short and broad, 11 bp per turn

B-DNA: right-handed, longer, thinner, 10 bp per turn

Z-DNA: left-handed, longest, thinnest, 12 bp per turn

Major and minor grooves are lined with sequence-specific H-bonding.

Consequences of double helical structure:

1. Facilitates accurate hereditary information transmission

2.Reversible melting• melting: dissociation of the double helix• melting temperature (Tm)• hypochromism• annealing

Supercoiling

relaxed DNA

supercoiled DNA

Tertiary Structure

Topoisomerase I – relaxation of supercoiled structures

Topoisomerase II – add negative supercoils to DNA

Structure of Single-stranded DNA

Stem Loop

Nucleic Acids: Structure

RNA

Secondary Structure

transfer RNA (tRNA) : Brings amino acids to

ribosomes during translation

Transfer RNA

Extensive H-bonding creates four double helical domains, three capped by loops, one by a stem

Only one tRNA structure (alone) is known

Many non-canonical base pairs found in tRNA

ribosomal RNA (rRNA) : Makes up the ribosomes, together with ribosomal proteins.

Ribosomes synthesize proteins

All ribosomes contain large and small subunits

rRNA molecules make up about 2/3 of ribosome

Secondary structure features seem to be conserved, whereas sequence is not

There must be common designs and functions that must be conserved

messenger RNA (mRNA) : Encodes amino acid sequence of a polypeptide

small nuclear RNA (snRNA) :With proteins, forms complexes that are used in RNA processing in eukaryotes. (Not found in prokaryotes.)

Central Dogma

DNA Replication, Transcription, and Translation

Central Dogma

DNA ReplicationCentral Dogma

DNA Replication – process of producing identical copies of original DNA

• strand separation followed by copying of each strand

• fixed by base-pairing rules

DNA replication is semi-conservative.

DNA replication is bidirectional.

involves two replication forks that move in opposite direction

DNA Replication

Begins at specific start sites• in E. coli, origin of replication, oriC locus• binding site for dnaA, initiation protein• rich in A-T

Overall: each of the two DNA duplexes contain one “old” and one “new” DNA strand (semi-conservative) and half of the new strand was formed by leading strand and the other half by lagging strand.

DNA replication requires unwinding of the DNA helix.

expose single-stranded templates

DNA gyrase – acts to overcome torsional stress imposed upon unwinding

helicases – catalyze unwinding of double helix- disrupts H-bonding of the two strands

SSB (single-stranded DNA-binding proteins) – binds to the unwound strands, preventing re-annealing

Primer

RNA primes the synthesis of DNA.

Primase synthesizes short RNA.

DNA replication is semidiscontinuous

DNA polymerase synthesizes the new DNA strand only in a 5’3’ direction. Dilemma: how is 5’ 3’ copied?

The leading strand copies continuously

The lagging strand copies in segments called Okazaki fragments (about 1000 nucleotides at a time) which will then be joined by DNA ligase

DNA Polymerase= enzymes that replicate DNA

All DNA Polymerases share the following:

1.Incoming base selected in the active site (base-complementarity)

2.Chain growth 5’ 3’ direction (antiparallel to template)3.Cannot initiate DNA synthesis de novo (requires primer)First DNA Polymerase discovered – E.coli DNA Polymerase I (by Arthur Kornberg and colleagues)

Roger D. Kornberg

2006 Nobel Prize in Chemistry

Arthur Kornberg

1959 Nobel Prize in Physiology and Medicine

http://www.nobelprize.org

DNA Polymerase

specificity dictated by H-bonding and shape complementarity between bases• binding of correct base is

favorable (more stable)• interaction of residues in

the enzyme to the minor groove of DNA

• close down around the incoming NTP

Mechanism of DNA linkage:

3’ 5’ exonuclease activity

- removes incorrect nucleotides from the 3’-end of the growing chain (proofreader and editor)- polymerase cannot elongate an improperly base-paired terminus

proofreading mechanisms• Klenow fragment – removes

mismatched nucleotides from the 3’’ end of DNA (exonuclease activity)

• detection of incorrect base- incorrect pairing with the template

(weak H-bonding)- unable to interact with the minor

groove (enzyme stalls)

5’ 3’ exonuclease activity

Exonuclease activity

- remove distorted segments lying in the path of the advancing polymerase

DNA Ligase = seals the nicks between Okazaki fragments

DNA ligase seals breaks in the double stranded DNA

DNA ligases use an energy source (ATP in eukaryotes and archaea, NAD+ in bacteria) to form a phosphodiester bond between the 3’ hydroxyl group at the end of one DNA chain and 5’-phosphate group at the end of the other.

DNA replication terminates at the Ter region.

• the oppositely moving replication forks meet here and replication is terminated

• contain core elements 5’-GTGTGTTGT

• binds termination protein (Tus protein)

Eukaryotic DNA Replication Like E. coli, but more complex

Human cell: 6 billion base pairs of DNA to copy

Multiple origins of replication: 1 per 3000-30000 base pairs

E.coli 1 chromosomeHuman 23E.coli circular chromosome; Human linear

Telomeres

The Ends of Linear DNA Possess TelomeresPresent because DNA is shortened after

each round of replicationContains hundreds of tandem repeats of a

hexanucleotide sequence (AGGGTT in humans)

Telomeres at the 3’ end is G rich and is slightly longer

May form large loops to protect chromosome ends

DNA Recombination =

recombinases Holliday junction –

crosslike structure

natural process of genetic rearrangement

Mutations

1. Substitution of base pair

a. transitionb. transversion

2. Deletion of base pair/s

3. Insertion/Addition of base pair/s

DNA replication error rate: 3 bp during copying of 6 billion bp

Macrolesions: Mutations involving changes in large portions of the genome

Agents of Mutations

1. Physical Agentsa) UV Lightb) Ionizing Radiation

2. Chemical AgentsSome chemical agents can be

classified further intoa) Alkylatingb) Intercalatingc) Deaminating

3. Viral

UV Light Causes Pyrimidine Dimerization

Replication and gene expression are blocked

Chemical mutagens

• 5-bromouracil and 2-aminopurine can be incorporated into DNA

Deaminating agentsEx: Nitrous acid (HNO2)Converts adenine to hypoxanthine, cytosine to uracil, and

guanine to xanthineCauses A-T to G-C transitions

Alkylating agents

Intercalating agents

AcridinesIntercalate in DNA, leading to insertion or

deletionThe reading frame during translation is changed

DNA Repair

Direct repairPhotolyase cleave pyrimidine dimers

Base excision repairE. coli enzyme AlkA removes modified bases

such as 3-methyladenine (glycosylase activity is present)

Nucleotide excision repairExcision of pyrimidine dimers (need different

enzymes for detection, excision, and repair synthesis)

Do we has a quiz?

QUIZ1. Draw the structure of any nitrogenous base of your

picking. (1 pt)

2. What is the difference between the glycosidic bond and the phosphodiester bond? (2 pts)

3. Give the reason why DNA utilizes the deoxyribose while RNA uses the ribose. (2 pts)

4. Enumerate all the enzymes and proteins involved in DNA replication and briefly state their importance/function. A short concise answer will suffice. (4 pts)

5. Give the partner strand of this piece of DNA:5-ACTCATGATTAGCAG-3 (1 pt)

RNA TranscriptionCentral Dogma

Process of Transcription has four stages:

1. Binding of RNA polymerase at promoter sites2. Initiation of polymerization3. Chain elongation4. Chain termination

Transcription (RNA Synthesis)

RNA PolymerasesTemplate (DNA)Activated precursors (NTP)Divalent metal ion (Mg2+ or Mn2+)

Mechanism is similar to DNA Synthesis

Reece R. Analysis of Genes and Genomes.2004. p47.

Limitations of RNAP II:

1. It can’t recognize its target promoter and gene. (BLIND)

2. It is unable to regulate mRNA production in response to developmental and environmental signals. (INSENSITIVE)

Start of Transcription

Promoter SitesWhere RNA Polymerase can indirectly bind

TATA box – a DNA sequence (5’—TATAA—3’) found in the promoter region of most eukaryotic genes.

Abeles F, et al. Biochemistry. 1992. p391.

Preinitiation Complex (PIC)

Transcription Factors (TF):

Hampsey M. Molecular Genetics of RNAP. Microbiology and Molecular Biology Reviews. 1998. p7.

TFIID binds to TATA; promotes TFIIB binding

TFIIA stabilizes TBP binding

TFIIB promotes TFIIF-pol II binding

TFIIF targets pol II to promoter

TFIIE stimulates TFIIH kinase and ATPase actiivities

TFII H helicase, ATPase, CTD kinase activities

Termination of Transcription

Terminator SequenceEncodes the

termination signalIn E. coli – base

paired hair pin (rich in GC) followed by UUU…

1. Intrinsic termination = termination sites

causes the RNAP to pause

causes the RNA strand to detach from the DNA template

Termination of Transcription

2. Rho termination = Rho protein, ρ

prokaryotes: transcription and translation happen in cytoplasm

eukaryotes: transcription (nucleus); translation (ribosome in cytoplasm)

In eukaryotes, mRNA is modified after transcriptionCapping, methylationPoly-(A) tailsplicing

capping: guanylyl residue

capping and methylation ensure stability of the mRNA template; resistance to exonuclease activity

Eukaryotic genes are split genes: coding regions (exons) and noncoding regions (introns)

Introns & Exons

IntronsIntervening

sequencesExons

Expressed sequences

Splicing

Spliceosome: multicomponent complex of small nuclear ribonucleoproteins (snRNPs)

splicing occurs in the spliceosome!

Reverse TranscriptionRNA-Directed DNA Polymerase

1964: Howard Temin notices that DNA synthesis inhibitors prevent infection of cells in culture by RNA tumor viruses. Temin predicts that DNA is an intermediate in RNA tumor virus replication

1970: Temin and David Baltimore (separately) discover the RNA-directed DNA polymerase - aka "reverse transcriptase"

Reverse Transcriptase

Primer required, but a strange one - a tRNA molecule that the virus captures from the host

RT transcribes the RNA template into a complementary DNA (cDNA) to form a DNA:RNA hybrid

All RNA tumor viruses contain a reverse transcriptase

RT II Three enzyme activities

RNA-directed DNA polymerase RNase H activity - degrades RNA in the DNA:RNA

hybrids DNA-directed DNA polymerase - which makes a

DNA duplex after RNase H activity destroys the viral genome

• HIV RT: very error-prone (1 bp /2000 to 4000 bp)

HIV therapy: AZT (or 3'-azido-2',3'- dideoxythymidine) specifically inhibits RT

Translation: Protein SynthesisCentral Dogma

TranslationStarring three types of RNA

1.mRNA

2.tRNA

3.rRNA

Properties of mRNA

1. In translation, mRNA is read in groups of bases called “codons”

2. One codon is made up of 3 nucleotides from 5’ to 3’ of mRNA

3. There are 64 possible codons

4. Each codon stands for a specific amino acid, corresponding to the genetic code

5. However, one amino acid has many possible codons. This property is termed degeneracy

6. 3 of the 64 codons are terminator codons, which signal the end of translation

Genetic Code

3 nucleotides (codon) encode an amino acid

The code is nonoverlappingThe code has no punctuation

Synonyms

Different codons, same amino acidMost differ by the last base

XYC & XYU XYG & XYA

Minimizes the deleterious effect of mutation

Encoded sequences. (a) Write the sequence of the mRNA molecule

synthesized from a DNA template strand having the sequence

(b) What amino acid sequence is encoded by the following base sequence of an mRNA molecule? Assume that the reading frame starts at the 5 end.

Practice

Answers

(a) 5’ -UAACGGUACGAU-3’ .(b) Leu-Pro-Ser-Asp-Trp-Met.

tRNA as Adaptor Molecules

Amino acid attachment site

Template recognition siteAnticodon

Recognizes codon in mRNA

tRNA as Adaptor Molecules

Mechanics of Protein Synthesis All protein synthesis involves three

phases: initiation, elongation, termination Initiation involves binding of mRNA and

initiator aminoacyl-tRNA to small subunit(30S), followed by binding of large subunit (50S) of the ribosome

Elongation: synthesis of all peptide bonds - with tRNAs bound to acceptor (A) and peptidyl (P) sites.

Termination occurs when "stop codon" reached

TranslationOccurs in the ribosomeProkaryote START

fMet (formylmethionine) bound to initiator tRNA

Recognizes AUG and sometimes GUG (but they also code for Met and Val respectively)

AUG (or GUG) only part of the initiation signal; preceded by a purine-rich sequence

Translation

Eukaryote START

AUG nearest the 5’ end is usually the start signal

Termination

Stop signals (UAA, UGA, UAG):• recognized by release factors (RFs)• hydrolysis of ester bond between polypeptide and

tRNA

Reference:

Garrett, R. and C. Grisham. Biochemistry. 3rd edition. 2005.

Berg, JM, Tymoczko, JL and L. Stryer. Biochemistry. 5th edition. 2002.