MCB2004C Chapter 8

What do chromosomes contain? This is known as the organisms' "blue print"

Double Stranded DNA

What does Adenine pair with?

Thymine

What does Cytosine pair with?

Guanine

True or False: DNA runs parallel

False

True or False: Bacterial and Eukaryotic genomes are organized the same

False

Intron

A noncoding region of a genome

Exon

A coding region of a genome

Bacterial Genome

Features one large coding region

Plasmids

Extra non-chromosomal piece of DNA found in bacteria.

Can exist alone outside of host DNA.

Bacteria can get rid of these if not needed.

Often contains genes that are useful for a specific environment.

May confer virulence genes.

Can introduce genes from unrelated species

True or False: Plasmids alter the species

False, but they can enter and leave cells

What dangerous pathogens can rise from the transfer of genes on plasmids

MRSA and MDR Mycobacterium (Multi Drug Resistance)

True or False: DNA replication is semi-conservative

True

When synthesizing a new strand of DNA, which way is it synthesized?

5' to 3'

Semiconservatism

New strand is synthesized by pairing with complementary base on the parent strand

DNA Polymerase

Helps add new bases to strand of DNA

Active Site

Makes sure the right base is being added

Helicase

An enzyme that will use energy to unwind DNA

What does the Helicase enzyme generate?

The replication fork

What are the 3 stages of DNA synthesis?

Initiation, Elongation, Termination

DNA Synthesis: Initiation

Begins at the origin (oriC)

5' to 3' synthesis.

Includes DNA polymerase, RNA primer.

Bidirectional

DNA Synthesis: Elongation

Bidirectional replication.

Replication bubble.

Produces the leading vs. lagging strand

DNA Synthesis: Termination

Replication forks meet at the terminus (ter site).

Methylation.

Negative supercoils

Summary of DNA Synthesis

1. Replication begins at origin.

2. Replication bubble forms, allowing the replication forks to progress in opposite directions.

3. Leading strand at each fork is synthesized continuously 5' to 3'.

4. Lagging strand at each fork is synthesized discontinuously in Okazaki fragments 5' to 3'.

5. Replication ends at terminus

True or False: Accuracy is important for DNA replication. DNA polymerase is always accurate.

False

DNA Polymerase I

Proofreads newly synthesized DNA and repairs and checks for correct bases.

Repair enzymes then detect mismatched pairs

Methylation

Allows for enzymes to detect the "new" strand

Transcription

The making of RNA by RNA Polymerase

Translation

The reading of mRNA and making it into protein(s) that begins at the start site

True or False: Bacterial cells replicate DNA and express genes simultaneously

True

Sigma Factor

Scans for particular genes (promoters and terminators).

Falls off allowing transcription to continue.

Looks at 45 bases/second

Steps of Transcription

1. RNA polymerase holoenzyme scans DNA for promoter sequences.

2. Binding to the promoter sequence forms the closed complex.

3. RNA polymerase unwinds DNA and begins transcribing RNA from ribonucleoside triphosphates (rNTPs).

4. Sigma Factor leaves the complex

Start Codon

AUG, codes for amino acid Methionine

Stop Codon

Three codons that do not specify an amino acid.

UAA, UAG, UGA

True or False: Bacterial ribosomes will begin translation before transcription is done

True

Transfer RNA (tRNA)

Reads and decodes codon and makes sure the correct Amino Acid sets are being added

Charged tRNA

Aminoacyl-tRNA syntheses binds correct amino acid to tRNA

Anticodon Loop

Exposes 3 unpaired bases and pairs them with the mRNA codon, allowing Amino Acids to be added to the peptide chain

What are ribosomes made up of in Translation?

30S and 50S Subunits

3 Stages of Translation

Initiation, Elongation, Termination

True or False: RNA Translation uses ATP

False, it uses GTP, which is another energy carrier

Translation: Initiation

30S subunit binds first to the mRNA.

tRNA initiates base pairs with start codon.

50S subunit joins complex secondly (this requires energy via GTP)

E Site

Exit Site

P Site

Peptide Site

A Site

Acceptor Site

Translation: Elongation

Aminoacyl tRNA binds to the A site.

50S subunit catalyzes peptide bond.

Elongation factor enters the A site and pushes tRNA over one pocket.

This is all due to the help of GTP Synthesis for energy

Translation: Termination

Terminates at stop codon, release factor enters A site.

Peptide is released; subunits fall off.

Chaperone proteins aid in folding of completed proteins

True or False: Replication of DNA is perfect

False, there is an error rate in some bacteria of about 1/10,000,000 base pairs

True or False: Mutations do not get repaired

True

Mutations

Permanent, heritable alteration in DNA sequences.

Harmful, beneficial (typically does not stick around), or neutral.

Codons

Groups of DNA bases form these that code for a specific Amino Acid to build proteins

Point Mutation/Base Substitution

A single change to one base

Silent Mutation

Variant of Point Mutation/Base Substitution

Change to a base that does not lead to anything

Missense Mutation

Variant of Point Mutation/Base Substitution

Base changes, causing a different Amino Acid to be coded

Nonsense Mutation

Variant of Point Mutation/Base Substitution

Base changes that makes a codon turn into the stop codon

Frameshift Mutation

Bases are either being added or removed

Inversion Mutation

A row of bases from the parental and newly synthesized DNA are flipped with one another and than the bases are reversed.

Example: TTTAA on top strand and AAATT on bottom strand. When this mutation happens, the bases become TTAAA on top, AATTT on bottom

What are the positive parts of having a mutation?

Defense against host immune system

What does mutation repair usually depend on?

Type of mutation requiring the repair and the extent of the damage.

Most organism have repair mechanisms in place to ensure accuracy

Base Excision Repair

Enzyme removes damaged base and DNA Polymerase I synthesizes a replacement

Methyl Mismatch Repair

Repair enzymes remove incorrect strand opposite of the methylated one and DNA Polymerase I fills in gaps

SOS Repair

NOT A TRUE REPAIR.

Introduces mutation and maintains circular chromosome but DOES NOT fix mutation

DNA Recombination

Crossing over and exchange of two DNA Helices

Steps for Base Excision Repair

1. DNA glycosylase binds to an excises the damaged base.

2. Endonuclease cleaves the phosphodiester backbone.

3. DNA Polymerase I cleaves old backbone and synthesizes replacement strand.

4. DNA ligase seals the DNA strand

Biotechnology

Analysis of DNA and construction of artificial DNA molecules for an applied purpose

What are some of the variety of tools available for biotechnology, both physical and chemical?

Electrophoresis.

Enzymes isolated from bacteria.

Plasmid Analysis.

DNA Hybridization.

PCR (Polymerase Chain Reaction).

DNA Sequence Analysis

cDNA

A DNA copy of mRNA, where mRNA is "reverse transcribed"

Reverse Transcriptase

Derived from retroviruses like HIV, this reads mRNA and makes DNA from it

True or False: Reverse Transcriptase is a way to copy protein-coding portion of genes, but it omits noncoding intron sequences

True

What can cDNA be cloned into?

A plasmid, which can then be transformed into a bacterium that can then express amounts of the protein.

Example: Insulin

Restriction Endonucleases

Plasmids that contain DNA sequences which are made to cut DNA

Where does the Restriction Endonucleases cut?

At the restriction enzyme site

What is the sequence considered that is cut by the Restriction Endonucleases?

Palindrome

Gel Electrophoresis

Technique that separates fragments of DNA.

Uses agarose, which forms solid gel at room temperature.

There are molecular holes.

When electricity is conducted, the DNA flows downwards to the positive pole.

The rate of movement depends on the size

True or False: DNA carries a positive charge

False