Lecture 7 - DNA & RNA Structures

Molecular genetics

The scientific study of the structure and function of genes at the molecular level

Central dogma

• An explanation of the flow of genetic information within a biological system

• first stated by Francis Crick

Central dogma (1970 restatement in Nature)

• Deals with the detailed residue-to-residue transfer of sequential information

• states that such information cannot be transferred back from protein to either protein or nucleic acid

Flow of genetic information in the central dogma

• DNA → (transcription) → RNA → (translation) → protein

• DNA also copies itself by replication

Who proposed the DNA double helix model and when?

James Watson and Francis Crick in 1953

DNA feature 1

Two polynucleotide chains wound in a right-handed (clockwise) double helix

DNA feature 2

The nucleotide chains are anti-parallel: one runs 5' → 3', the other 3' → 5'

DNA feature 3

Sugar-phosphate backbones are on the outside of the double helix; the bases point toward the central axis

DNA feature 4

Complementary base pairs from opposite strands are held together by weak hydrogen bonds: A pairs with T (2 H-bonds), G pairs with C (3 H-bonds)

DNA feature 5

• Base pairs are 3.4 Å apart

• one complete turn of the helix is 36 Å (about 10 bases per turn)

DNA feature 6

The sugar-phosphate backbones are not equally spaced, creating major and minor grooves

Four famous experiments in the search for genetic material

Griffith's transformation experiment, Avery's DNA experiment, Hershey-Chase bacteriophage experiment, tobacco mosaic virus (TMV) experiment

Griffith's transformation experiment

Discovered the "transformation principle"

Avery's DNA experiment

Showed that DNA (not RNA) is the genetic material

Hershey-Chase bacteriophage experiment

Showed that DNA (not protein) is the genetic material

Tobacco mosaic virus (TMV) experiment

Showed that RNA can be a genetic material

Chargaff's Rule 1

The amount of adenine approximately equals thymine, and the amount of guanine approximately equals cytosine (so purines = pyrimidines)

Chargaff's Rule 2

The composition of DNA varies from one species to another

Rosalind Franklin

• Worked with Maurice Wilkins at King's College London

• used X-ray diffraction to study DNA fibers and made major advances in X-ray diffraction of DNA (1920-1958)

How is an X-ray diffraction pattern used?

It is interpreted using mathematical theory, which can reveal the structure of the molecule

Three DNA features suggested by Franklin's diffraction pattern

1) Helical

2) More than one strand

3) 10 base pairs per complete turn

Nucleotide

The unit (building block) of DNA

Three components of a DNA nucleotide

1) Deoxyribose (sugar)

2) Nitrogenous base

3) Phosphate

Two types of nitrogenous bases

Purines and pyrimidines

Purines

Adenine (A) and Guanine (G); double-ring structure

Pyrimidines

Cytosine (C) and Thymine (T) in DNA (Uracil in RNA); single-ring structure

dNTPs

• Deoxynucleoside triphosphates (dATP, dTTP, dCTP, dGTP)

• the building blocks for DNA synthesis

Energy source for building the DNA backbone

Hydrolysis of dNTPs, which releases energy and pyrophosphate (PPi)

When are dNTPs required?

For both in vivo and in vitro (e.g., PCR) DNA synthesis

Phosphodiester linkage

The covalent bond linking one nucleotide to the next in the DNA (and RNA) backbone

Direction of DNA

Read 5' to 3': starts at the phosphate group (5' end) and runs toward the sugar end (3' end)

5' end vs 3' end of a DNA strand

5' end: phosphate group attached to the 5' carbon of the sugar

3' end: hydroxyl (OH) group on the 3' carbon of the sugar

Where do bases attach in a nucleotide?

• To the sugar (deoxyribose)

• the bases project out from the sugar-phosphate backbone

DNA strands as templates

Watson and Crick proposed that each strand serves as a template for making a new strand, so DNA can be copied over and over (life requires this)

How are DNA strands copied?

By complementary base pairing: the molecule opens up and bases match with their complements (A with T, C with G)

DNA supercoiling

Over- or under-winding of DNA strands

Why is DNA supercoiling important?

• DNA can be thousands of times longer than the cell

• supercoiling reduces the space it takes and allows much more DNA to be packaged

Bacterial chromosome compaction

• Circular DNA → loop domains formed with associated proteins → supercoiled, looped DNA

• compacted about 1000-fold to fit in the cell

Three DNA conformations

A-DNA, B-DNA, and Z-DNA

B-DNA

• The most common conformation in most living cells

• the structure proposed by Watson and Crick

• right-handed

A-DNA

• A shorter, wider, right-handed helix

• found in dehydrated DNA samples and rarely under normal physiological conditions

Z-DNA

• A left-handed helix (unlike A- and B-DNA)

• a transient form that exists only occasionally in response to certain biological activity

Discovery and role of Z-DNA

• Discovered in 1979

• certain proteins bind it very strongly, suggesting it plays a role in protection against viral disease

Open reading frame (ORF)

A span of DNA sequence between a start codon and a stop codon

In how many directions can DNA be read for protein synthesis?

Only one direction

Sense strand

• The DNA strand that, read in the right direction, provides the code for a protein

• same sequence as the mRNA (with T instead of U); also called the coding strand

Antisense strand

• The DNA strand bonded to the sense strand

• also called the noncoding strand

• it is the template for mRNA synthesis

Relationship between mRNA and the DNA strands

mRNA is made from the antisense (template) strand and has the same sequence as the sense strand (U replaces T)

PCR

Polymerase chain reaction: a technique to make many copies of a specific DNA region in vitro (in a test tube rather than an organism)

Taq polymerase

The thermostable (heat-resistant) DNA polymerase used in PCR

Primers

• Short single-stranded DNA oligonucleotides designed specifically for the DNA region of interest

• they bind the template and give the polymerase a starting point

How does PCR make many copies?

The reaction is repeatedly cycled through a series of temperature changes

Common uses of PCR

DNA cloning, medical diagnostics, and forensic DNA analysis

Copies after 30 cycles of PCR

About 1.07 billion (2³⁰), because each cycle doubles the DNA

Five ingredients of a PCR mixture

1) dsDNA template

2) a pair of single-stranded DNA primers

3) heat-resistant DNA polymerase

4) dNTPs (dATP, dCTP, dGTP, dTTP)

5) buffer and cofactors (MgCl2)

PCR step 1: Denaturation

• 96-98°C (diagram shows 94°C) for 30-60 seconds

• heat separates the DNA strands, giving single-stranded templates

PCR step 2: Annealing

• 55-65°C (about 5°C below the primer Tm)

• cooling lets primers bind their complementary sequences on the template

PCR step 3: Extension

• 72°C

• Taq polymerase extends the primers, synthesizing new DNA strands

Order of PCR steps

Denaturation → Annealing → Extension (then repeat)

Three categories of PCR applications

Molecular identification, sequencing, and genetic engineering

RNA

• Ribonucleic acid: a polymer of ribonucleotides linked by phosphodiester linkages

• usually single-stranded and helical

Three components of an RNA nucleotide

1) Ribose (sugar)

2) Nitrogenous base (A, U, G, C)

3) Phosphate group

DNA sugar vs RNA sugar

DNA: deoxyribose (H at the 2' carbon)

RNA: ribose (OH at the 2' carbon)

DNA bases vs RNA bases

DNA: A, T, G, C

RNA: A, U, G, C (uracil replaces thymine)

Nucleoside

Base + sugar (e.g., adenine + ribose = adenosine)

Nucleotide (definition)

Base + sugar + phosphate(s) (e.g., AMP, ADP, ATP)

AMP, ADP, ATP

Adenosine monophosphate (1 phosphate), adenosine diphosphate (2), adenosine triphosphate (3)

RNA vs DNA structure

Structurally similar, but DNA is typically long and double-stranded, while RNA is much shorter and typically single-stranded

Main role of RNA

A variety of roles, but mainly protein synthesis (translation) and its regulation

How does single-stranded RNA form a 3D structure?

It folds on itself, and the folds are stabilized by short regions of complementary base pairing within the molecule

Two main classes of RNA

mRNA (messenger RNA) and ncRNA (non-coding RNA)

Two classes of non-coding RNA

Housekeeping ncRNA and regulatory ncRNA

Housekeeping ncRNAs

tRNA (transfer RNA) and rRNA (ribosomal RNA)

Two classes of regulatory ncRNA

lncRNA (long non-coding RNA, 200 or more nucleotides)

small ncRNA (fewer than 200 nucleotides)

Types of small ncRNA

microRNA (19-22 nucleotides)

snoRNA (small nucleolar RNA)

siRNA (small interfering RNA)

snRNA (small nuclear RNA)

piRNA (PIWI-interacting RNA)

mRNA

Single-stranded RNA that corresponds to a gene's DNA sequence and is read by a ribosome to make a protein (translation)

How much of a cell's total RNA is mRNA?

About 5%

Polycistronic mRNA

• mRNA that encodes more than one protein, with multiple translation start sites

• typical of many bacterial and chloroplast mRNAs

Monocistronic mRNA

• mRNA that encodes only one protein, with a single translation start site

• all eukaryotic mRNAs are monocistronic

Prokaryotic vs eukaryotic mRNA structure

Prokaryotic: 5' UTR, several protein-coding regions, 3' UTR; Eukaryotic: 5' m7G cap, UTR, one protein-coding region, UTR, 3' poly-A tail (AAAA(n))

Two special structures of eukaryotic mRNA

5' cap and 3' poly-A tail

Parts of a eukaryotic mRNA (5' to 3')

Cap → 5' UTR (leader) → coding sequence (start codon AUG to stop codon, e.g., UAG) → 3' UTR (trailer) → poly-A tail

5' cap

7-methylguanosine triphosphate added to the 5' end of the mRNA

Capping

Adding a methyl group (methylated guanosine cap) to the 5' end of the transcribed RNA

Two functions of the 5' cap

1) Protects against degradation by 5' exonucleases

2) Lets the cell's machinery (e.g., nuclear export proteins and ribosomes) recognize the transcript

Polyadenylation

Adding a long chain of adenine nucleotides (poly-A tail) to the 3' end of the transcript

Length of the poly-A tail

About 100-200 adenine nucleotides

Functions of the poly-A tail

Protects the mature mRNA, improves its stability, and helps its export from the nucleus

rRNA

• Ribosomal RNA: the RNA component of ribosomes

• essential for protein synthesis in all living organisms

Functions of rRNA

Not fully understood, but necessary for ribosome assembly and seems to play key roles in binding mRNA to ribosomes and translating it

Composition of ribosomes by weight

About 60% rRNA and 40% protein

Ribosome contents

Two major rRNAs and 50 or more proteins

Prokaryotic rRNAs and their eukaryotic equivalents

• Prokaryotic 23S, 16S, and 5S are equivalent to eukaryotic 28S, 18S, and 5.8S

• both come from processing a single pre-rRNA transcript

Prokaryotic 70S ribosome

Large 50S subunit (23S and 5S rRNAs, 34 proteins) + small 30S subunit (16S rRNA, 21 proteins)

Eukaryotic 80S ribosome

Large 60S subunit (28S, 5.8S, and 5S rRNAs, about 45 proteins) + small 40S subunit (18S rRNA, about 30 proteins)

"S" in 16S rRNA

Sedimentation coefficient: reflects how fast a macromolecule moves downward when centrifuged

tRNA

• Transfer RNA: the smallest of the three major types of RNA (74-95 nucleotides)

• transfers amino acids from the cytoplasm to the ribosome

How many kinds of tRNA are there?

At least 20, one for each of the 20 amino acids used in protein synthesis

tRNA 3' end

Ends in CCA, with the amino acid attached to the terminal A (A-OH)

tRNA primary structure

The nucleotide sequence, which allows extensive base pairing within the strand to form a secondary structure