Molecular Biology

• Molecular biology is the study of DNA– Its structure– How it replicates (and assembles to create

genetically-distinct offspring)– How it controls the cell by directing RNA and

protein synthesis• How does DNA store genetic information,

copy it, and pass it along from one generation to the next?

DNA and RNA

• DNA and RNA are nucleic acids consisting of long chains of nucleotides (collectively called a polynucleotide)

• There are 4 types of nucleotides that make up DNA, each with a different nitrogenous base– Adenine (A)– Cytosine (C)– Thymine (T)– Guanine (G)

Sugar-phosphate backbone

DNA nucleotide

Phosphate group

Nitrogenous baseSugar

DNA polynucleotide

DNA nucleotide

Sugar(deoxyribose)

Thymine (T)

Nitrogenous base(A, G, C, or T)

Phosphategroup

DNA and RNA

• RNA has the nitrogenous base Uracil (U), instead of Thymine (T), and is usually single-stranded

• DNA is double-stranded and forms a double helix

• The 2 sugar-phosphate backbones that form the double helix run in opposite directions (5’ to 3’ and 3’ to 5’)

Hydrogen bond

Basepair

Partial chemical structure Computer modelRibbon model

Each strand of DNA runs in opposite directions

DNA replication depends on specific base pairing

• The specific pairing of bases in DNA is evidence for a copying mechanism for the genetic material

• Knowledge of the sequence of bases in 1 strand of DNA allows you to determine the sequence in the second strand

• When 2 strands of DNA separate, each strand serves as a template for the assembly of a complimentary strand

DNA Replication

• The human genome (all genes collectively) contains over 6 billion base pairs in 46 chromosomes (23 ‘homologou’s pairs)!

• Yet, DNA replication requires only a few hours and is astonishingly accurate

• How does this process occur and what controls it???

DNA Replication

• DNA replication requires more than a dozen enzymes and other proteins (of course!)

• Replication of DNA begins at specific sites called origins of replication

• Origins of replication consist of a specific sequence of nucleotides where proteins attach to the DNA and separate the strands

• Replication then proceeds in both directions

Origin of replication Parental strand

Daughter strand

Bubble

Two daughter DNA molecules

DNA Replication

• Eukaryotic DNA has many origins of replication shortening the overall time needed for the replication process

• Replication occurs in “bubbles” of parental (old) and daughter (new) DNA

• Eventually, all the “bubbles” merge yielding 2 completed daughter strands of DNA

Daughter strands (grey); Parental strands (blue)

DNA Replication

• The enzymes that link DNA nucleotides to a growing daughter strand are called DNA polymerases

• Remember that DNA’s sugar-phosphate backbones run in opposite directions

• DNA polymerases add nucleotides only to the 3’ end, never to the 5’ end

• Thus, a daughter strand grows from 5’ to 3’ (Say what?!!?)

3 end 5 end

3 end5 end

3

5

2

4

13

5

2

41

P

P

P

P

P

P

P

P

Parental DNA

35

DNA polymerasemolecule

Daughter strandsynthesizedcontinuously

35

35

Daughter strandsynthesizedin pieces

• Since the 2 DNA strands run in opposite directions, and replication always begins at the 3’ end, the new daughter strand will be laid down beginning at its 5’ end

• 1 daughter strand is synthesized continuously, while the other must work outward from the forking point

• The new strand is synthesized in short pieces as the DNA strand opens up

• Another enzyme, called DNA ligase then links the pieces together to form a single DNA strand

Formed 1st Formed 2nd

Formed last

Thank you, polymerases

• DNA polymerases also carry out a proof-reading step to quickly remove any nucleotides that have been paired incorrectly during replication

• DNA polymerases and ligases are also involved in repairing DNA damaged by harmful radiation or toxic chemicals, including those found in cigarette smoke!

DNA Replication

• DNA replication ensures that all cells in a multicellular organism carry the same genetic information

• DNA replication occurs during interphase!• The DNA genotype is expressed as proteins,

which provides the molecular basis for phenotypic traits– DNA dictates the synthesis of proteins which

determine the traits physically expressed by an organism

DNA is transcribed into RNA and translated into Protein

• A gene does not build a protein directly• Instead, a gene dispatches its instructions for

building proteins in the form of RNA, which in turn directs protein synthesis

• The transcription of DNA into RNA and the subsequent translation of RNA into proteins is considered the “central dogma” of molecular biology

Cytoplasm

Nucleus

DNA

Transcription of DNA into RNA

RNA

Translation of RNA into Protein

Protein

DNA is life…the rest is just translation

• In eukaryotic organisms, DNA is stored in the nucleus where it is transcribed into RNA; a process called transcription

• RNA translates the information from DNA into proteins in the cytoplasm (or to be more precise, in the ribosomes… we’ll come back to this); a process called translation

Genetic information written in codons is translated into amino acid sequences

• A typical gene consists of hundreds or thousands of nucleotides in a specific sequence

• The sequence (and number) of these nucleotides determines the protein produced by this gene, and hence its resulting phenotype

• DNA must first be re-written (transcribed) as a sequence of RNA

Genetic information written in codons is translated into amino acid sequences

• Translation then converts the nucleic acid ‘language’ into the polypeptide (protein) ‘language’

• The sequence of RNA nucleotides dictates the sequence of amino acids of the polypeptide being produced

• Thus, the RNA molecule acts as a messanger carrying genetic information from DNA

Polypeptide

Translation

Transcription

DNA strand

Codon

Amino acid

RNA

Genetic information written in codons is translated into amino acid sequences

• In order for translation to proceed, the sequence of the 4 nucleotides in RNA (A,U, C,G) must somehow specify the 20 amino acids used to make up proteins

• The flow of information from gene to protein is based on a triplet code; genetic instructions for the amino acid sequences of a polypeptide chain are written in DNA and RNA as a series of 3-base ‘words’, called codons

The Genetic Code

• The genetic code is a set of instructions indicating which codons are translated into which amino acid

• The genetic code does not only specify which codons code for which amino acids, but also specify ‘start’ and ‘stop’ signals, which begin and end protein synthesis, respectively

• For each of the 20 amino acids, there are 2-4 codons which code exclusively for them

Firs

t bas

e

Third

bas

e

Second base

The Genetic Code

• The genetic code is nearly universal; humans cells can translate bacterial RNA and vice versa

Transcription

• An enzyme called RNA polymerase attaches to an area of one of the DNA molecules in the double helix and moves along the DNA strand ‘reading’ the nucleotides

• It then selects complimentary nucleotides and links them one by one via hydrogen bonds

• A nucleotide sequence called a promoter serves as a “start” signal, while a terminator sequence marks the end of transcription

TerminatorDNA

DNA of gene

RNA polymerase

Initiation

PromoterDNA

1

Elongation2

Area shownin Figure 10.9A

Termination3

GrowingRNA

RNApolymerase

CompletedRNA

RNApolymerase

Newly made RNA

Direction oftranscription Template

strand of DNA

RNA nucleotides

Messenger RNA

• The type of RNA that encodes amino acid sequences is called messenger RNA (mRNA)

• In eukaryotic cells, mRNA leaves the nucleus where it had been transcribed and enters the cytoplasm

• Before mRNA can leave the nucleus, it is modified– A ‘tail’ and ‘cap’ are added– Introns are removed

RNAtranscriptwith capand tail

Exons spliced together

Introns removed

TranscriptionAddition of cap and tail

Tail

DNA

mRNA

Cap

Exon Exon ExonIntron Intron

Coding sequenceNucleus

Cytoplasm

Introns are intervening sequences of DNA which do not code for amino acids; must be removed

Exons are the coding regions, parts of the gene which remain and are translated into amino acids

A cap and tail are added to protect the mRNA strand, facilitate its transport out of the nucleus and to help ribosomes bind to it

Transfer RNA

• In order to convert the 3-letter codons of nucleic acids into a single amino acid, a cell must employ a molecular interpreter, transfer RNA (tRNA)

• tRNA recognizes the codons in the mRNA molecule and picks out the appropriate amino acids for incorporation into the growing polypeptide

Transfer RNA

• tRNA recognizes codons from mRNA via a special triplet of bases called an anticodon, which is complimentary to the codon on the mRNA

• When the codon of mRNA complements the anticodon of tRNA, the appropriate amino acid is laid down at the other end of the tRNA molecule

Anticodon

Amino acid attachment site

RNA polynucleotide chain

Hydrogen bond

Transfer RNA

• For each amino acid, there is a specific type of tRNA that it will bind to

• And for each tRNA, there is a specific enzyme which binds the amino acid to its specific tRNA molecule

• How many enzymes (or tRNA molecules for that matter) are there?

Translation

• mRNA leaving the nucleus enters the cytoplasm where it binds to a ribosome (Remember, all cells contain ribosomes…)

• Translation begins when the mRNA molecule arrives at the ribosome

• While mRNA was being synthesized, tRNA molecules were already uniting with their specific amino acids

Translation

• The tRNA molecules then begin transporting their amino acids to the ribosomes to meet the mRNA molecule

• Ribosomes are made up of proteins and a type of RNA called ribosomal RNA (rRNA)

• The ribosomes contain binding sites for both mRNA and tRNA

tRNA-binding sites

Largesubunit

Smallsubunit

mRNAbinding site

mRNA

Next amino acidto be added topolypeptide

Growingpolypeptide

Codons of mRNA

tRNA

Anticodon of tRNA

New peptidebond formingGrowing

polypeptide

4

A succession of tRNAsadd their amino acidsto the polypeptide chainas the mRNA is movedthrough the ribosome,one codon at a time.

Elongation

Codons

mRNA

Polypeptide

5

The ribosome recognizes a stop codon. The polypeptide is terminated and released.

Termination

Stop codon

Translation

• Translation begins with a ‘start’ codon, and ends with a ‘stop’ codon

• The amino acid methionine (Met) is always translated by the start codon (AUG)

• What would the anticodon look like?• Stop codons (UAA, UAG, and UGA) do not

code for amino acids but instead act only as signals to end translation

Review

• Describe the differences between mRNA, tRNA and rRNA

• What bases are found in DNA? In RNA?• Which molecule has codons? Anticodons?• What is transcription? Translation? Which

happens first and where does each occur in the cell?

Mutations• A single change in the amino acid coded for by

a gene can lead to mutation• …and a single change to a single nucleotide

can lead to a change in amino acid!• Mutations can be caused

by a nucleotide addition, deletion or substitution

• Insertions or deletions are the most disastrous

www.milehive.com/.../x-men-origins-wolverine.jpg

Mutations

• The production of mutations can occur spontaneously during DNA replication or by a mutagen, a physical or chemical agent such as X-rays and ultraviolet light (physical)

• What would happen if a mutation occurred in an intron? An exon?

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Viruses

• A virus is a fragment of nucleic acid surrounded by a protein coat

• Viruses are infectious; they are parasites that can reproduce only inside living cells

• The host cell provides most of the components necessary for replicating, transcribing and translating the viral DNA!

You can run, but you can’t hide…

• Viruses infect bacteria, archaea, protists, plants and animals, and are found in nearly every ecosystem on Earth!

• Viruses contain genes made of DNA or RNA

• The protein coat (or membrane in some cases) allows the virus to penetrate the host cell

Viral DNA

Viruses

• Viruses cause illness because they attach to a cell, and inject their DNA into it

• The host cell is then ‘instructed’ by the viral DNA to produce more copies of itself and to translate proteins, which together serve to assemble more viruses!

• Eventually the cell lyses and releases an army of viruses

Phage attachesto bacterial cell.

Phage injects DNA. Phage DNA directs hostcell to make more phageDNA and protein parts.New phages assemble.

Cell lyses andreleases new phages.

Lytic cycle of viruses

Viruses

• The common cold is caused by viruses containing RNA, as are measles, mumps, AIDS and polio

• DNA viruses cause hepatitis, chicken pox and herpes

• Glycoproteins on the virus’s outer coat enable it to attach to receptor proteins on the host cell’s plasma membrane (very specific!)

Got NyQuil?

• The amount of harm caused by viruses depends largely on how quickly an organism’s immune system responds to fight the infection, and also on the ability of the infected tissue to repair itself

• Our respiratory tract can efficiently replace damaged cells by mitosis and we usually recover quickly from colds, but damage done to nerve cells by the Poliovirus is permenant

How do viruses spread?

• Ever wonder why we sneeze and cough when we’re sick???

• Cold sores, herpes, chicken pox…..

Viral DNA may become part of the host chromosome

• Viruses reproduce via the host cell as previously described in the lytic cycle

• But viruses can also reproduce via an alternative route called the lysogenic cycle

• During a lysogenic cycle, viral DNA is replicated without destruction of the host cell

• In this case, viral DNA is incorporated into the host cell’s DNA and is replicated every time the host cell prepares to divide

Bacterialchromosome

Phage injects DNA

Phage

Phage DNA

Attachesto cell

2

1

3

Phage DNAcircularizes

Lytic cycle

4

New phage DNA andproteins are synthesized

Phages assemble

Cell lyses,releasing phages

65

7

Phage DNA inserts into the bacterialchromosome by recombination

Lysogenic bacterium reproducesnormally, replicating theprophage at each cell division

Prophage

Lysogenic cycle

Many celldivisions

OR

Lytic and Lysogenic viral cycles

Viruses

• The bacteria that cause diphtheria, botulism and scarlet fever would be harmless were it not for the viral DNA encoded into their DNA!

• Mutations of existing viruses are a constant source of new, emerging viruses

• RNA viruses are usually the culprit; errors in replication are not subject to the types of proofreading mechanisms that help reduce mutations in DNA replication

Question of the day (or century, millennium, etc ….)

• Are viruses alive????• Do they reproduce?• Do they grow and develop?• Do they take in energy and process it to

perform their activities?• Do they respond to their environment?• Do they adapt?

Want to learn more?