Pre Med III Genetics

Guri Tzivion, PhD

tzivion@windsor.eduExtension 506

Summer 2015Windsor University School of Medicine

Pre Med III GeneticsClass 14

Review for Block 2 exam

Review for Block 2 exam

The exam will cover:

RNA & Transcription

Proteins and Translation

Mutations and DNA Repair

Regulation of Gene Expression:

Prokaryotes & Eukaryotes

Recombinant DNA & Molecular Biology Methods

RNA & Transcription

DNA RNA protein

transcription translation replication

reverse transcription

Central dogma

MAJOR CLASSES OF RNA

rRNA (ribosomal RNA) mRNA (messenger RNA) tRNA ( transfer RNA)

snRNA (small nuclear RNA)hn RNA (heterogeneous nuclear

RNA) miRNA (micro RNA)

RNA V/S DNA

replication transcription

template double strands single strand

substrate dNTP NTP

primer yes no

Enzyme DNA polymerase RNA polymerase

product dsDNA ssRNA

base pair A-T, G-C A-U, T-A, G-C

Differences between replication and transcription

Transcription in Bacteria

mRNA transcript is synthesized by RNA polymerase in the 5’ to 3’ direction.

Template strand:DNA strand that is read (transcribed) by RNA

polymerase.

Non-template strand:DNA strand that is not read by RNA polymerase.

Transcription initiation

• 1. Sigma binds to specific promoter regions of DNA (-35 box and -10 box).

Phase 3: Transcription termination

• RNA polymerase encounters a termination signal within the DNA template, which codes for a hairpin loop structure in the RNA.

• The hairpin causes the RNA polymerase to separate from the RNA transcript, ending the transcription.

DNA

Cytoplasm

Nucleus

Eukaryotic Transcription

ExportG AAAAAA

RNA

Transcription

Nuclear pores

G AAAAAA

RNAProcessing

mRNA

Eukaryotic mRNA Processing

• After transcription, specific regions of the primary RNA transcript are spliced out and degraded during RNA processing.

• Exon: Contains sequences required for protein synthesis and is NOT spliced out during RNA processing. Is part of the final mature spliced RNA transcript.

• Intron: Spliced out during RNA processing and degraded.

Exon 1 Exon 2 Exon 3

Eukaryotic mRNA Processing

• Besides splicing, other steps involved in mRNA processing include:

1. Addition of a “cap” at the 5’ end of the mRNA. The “5’ cap” serves as a recognition signal for the translation machinery of the cell.

2. Addition of poly (A) tail at the 3’ end of the mRNA. Serves to stabilize the mRNA by protecting it from enzymatic degradation.

Proteins and Translation

From messenger RNA to protein: Translation

The mRNA is translated by ribosomes as series of 3 letter codes designated codons

Second mRNA base

Fir

st m

RN

A b

a se

(5¢

e nd

)

Th

ird

mR

NA

bas

e (3

¢ en

d)

The Genetic Code

The Genetic Code

General tRNA structure: ~ 80 nt long

Met

GTPInitiator tRNA

mRNA

5¢3¢

mRNA binding site

Smallribosomalsubunit

Start codon

P site

5¢3¢

Translation initiation complex

E A

Largeribosomalsubunit

GDP

Met

• The initiation process involves the association of the mRNA, the initiator methionine-tRNA and the small ribosomal subunit. Several additional “initiation factors” -are also involved. The large ribosomal subunit then joins the complex.

Initiation in prokaryotes

Schematic model showing the binding sites on the ribosome

P site (Peptidyl-tRNAbinding site)

E site (Exit site)

mRNAbinding site

A site (Aminoacyl-tRNA binding site)

Largesubunit

Smallsubunit

E P A

The assembled ribosome has one exit site and two tRNA-binding sites, which are called A- and P-site, for aminoacyl and peptidyl sites respectively.

Only fMet-tRNAfMet can be used for initiation by 30S subunits; all other aminoacyl-tRNAs are used for elongation by 70S ribosomes.

Polypeptide

tRNA withamino acidattached

Ribosome

tRNA

Anticodon

3¢5¢

mRNA

Aminoacids

Codons

Elongation

- Amino acids are added one by one to the preceding amino acid

-Elongation factors facilitate

- codon recognition

- peptide bond formation

- translocation

Ribosome ready fornext aminoacyl tRNA

mRNA

5¢

Amino endof polypeptide

E

Psite

Asite

3¢

2

2 GDP

E

P A

GTP

GTP

GDP

E

P A

E

P A

1. Recognition

2. Peptide bondformation3. Translocation

3¢

The release factor hydrolyzes thebond between the tRNA in theP site and the last amino acid of thepolypeptide chain. The polypeptideis thus freed from the ribosome.

The two ribosomal subunitsand the other componentsof the assembly dissociate.

Releasefactor

Stop codon(UAG, UAA, or UGA)

5¢

3¢

5¢

3¢

5¢

Freepolypeptide

When a ribosome reaches a stopcodon on mRNA, the A site of theribosome accepts a protein calleda release factor instead of tRNA.

Termination

Mutations and DNA Repair

Mutations, definition

• Mutation - any change made to the DNA sequence or chromosome structure.

1) Inherently can either have beneficial or negative effect or have no significance. For example, they can lead to disease or death or promote evolution by generating new alleles.

2) They are permanent – can’t be removed or repaired (damage versus mutation)

3) They do not occur selectively and are random

3) The type of the cell that contains the mutated DNA:

a) Somatic mutations, arise in the DNA of somatic cells (normal diploid cells), do not pass

to the next generation. b) Germ-line mutations: arise in the DNA of gamete-

forming tissue (those cells that produce sperm and eggs). Are transmitted to the offspring and pass to the future generations.

• Small gene mutations come in 3 main varieties:

A. Base-pair substitutions: One nucleotide is changed to a different nucleotide.

Three possible outcomes on the amino acid sequence: 1. Silent mutation: No effect, usually when the change

occurs in the 3rd nucleotide of a codon.

2. Missense mutation: The change causes the wrong amino acid to be inserted. Can be Natural mutation if the new amino acid has a similar structure to the previous aa.

3. Nonsense mutation: Change turns the codon into a stop codon. Results in a truncated protein.

A. Base-pair substitutions:

1. Silent mutation: No effect, usually when the change occurs in the 3rd nucleotide of a codon.

2. Missense mutation: The change causes the wrong amino acid to be inserted. Can be Natural mutation if the new amino acid has a similar structure to the previous aa.

3. Nonsense mutation: Change turns the codon into a stop codon. Results in a truncated protein.

B. Insertion/deletion: An extra nucleotide gets added or removed, causing a frame-shift. All amino acids after the insertion/deletion site will be altered!!

Excision repairTwo major types of excision repair:

I. Base-Excision repair: Remove abnormal or modified bases from DNA.

II. Nucleotide-Excision Repair: Remove larger defects like thymine dimers.

Base- Excision Repair:

Initiated by a group of enzymes called DNA glycosylases (recognize abnormal bases in DNA).

The glycosylases cleave glycosidic bonds between the abnormal base and the 2-deoxyribose.

Base Excision Repair

There are different DNA glycosylases, for different types of damaged bases.

AP endonuclease recognizes sites with a missing base; cleaves sugar-phosphate backbone.

Deoxyribose phosphodiesterase removes the sugar-phosphate lacking the base.

Nucleotide Excision Repair

Base excision repair Nucleotide excision repair

Mismatch repair system

Sample Questions

1. Mutations:

a. Are permanent changes in the DNA sequence or structure.

b. Produce allelic variation.

c. Are more likely to be harmful than beneficial.

d. All of the above.

e. None of the above.

Sample Questions

1. Mutations:

a. Are permanent changes in the DNA sequence or structure.

b. Produce allelic variation.

c. Are more likely to be harmful than beneficial.

d. All of the above.

e. None of the above.

Regulation of Gene

Expression

Prokaryotes & Eukaryotes

Regulation of Gene Expression

Gene expression can be regulated During transcription (transcriptional control). During translation (translational control). After translation (post-translational control).

Positive Control of Transcription

Positive control occurs when a regulatory protein (activator) binds to DNA and increases the rate of transcription of downstream genes.

Negative Control of Transcription

Negative control occurs when a regulatory protein (repressor) binds to DNA and decreases the rate of transcription of downstream genes.

Copyright © 2006 by Elsevier, Inc.

The Operon: a procaryote model• series of genes and their shared regulatory elements

• gene products contribute to a common process

Transcriptional Control:

The lac and trp operons

Glucose regulates cAMP levels

Positive control of the lac operon

When cAMP levels are high, CAP is activated, inducing its binding to the CAP site and promoting efficient transcription.

Summary of the lac operon

Examples of gene expression regulation

Transcriptional control:Regulatory proteins affect the ability for the RNA polymerase to bind to or transcribe a particular gene.

Translational control:Regulatory proteins can affect the rate of translation.Enzymes can affect the stability of the mRNA.

Post-translational control:Translated protein may be modified by phosphorylation or other modifications that alter the protein’s activity, folding or stability.

AP Biology

Points of control The control of gene

expression can occur at any step in the pathway from gene to functional protein1. Packing/unpacking of DNA

2. Transcription

3. mRNA processing

4. mRNA transport

5. Translation

6. Protein processing

7. Protein degradation

• Histone deacetylases (HDACs) are negative regulators of transcription

• Histone acetylases (HATs) are positive regulators of transcription

In general…

+

-

CBP/P300

HDAC1/2

DNA methylation• DNA can be modified by methylation of adenine and cytosine bases

Methylated Base Methylation Sequence

C5-methylcytosine (5-mC) CpG

C5-hydroxymethylcytosine (5-hmC)

CpG, CpHpG1, CpHpH1

H = Adenine, Cytosine, or Thymine

"p" in CpG refers to the phosphodiester bond

Components

of epigenetic

inheritence

Transcriptional regulatory elements in eukaryotes

Control of transcription: promoters, enhancers, repressors….

Control of transcription in eukaryotes depends on both cis- and trans-acting factors, including: regulatory & promoter sequences, RNA polymerase, the general transcription factors and gene specific activators and repressors

Cis- versus trans-acting factors

Cis: includes all the elements that are present on the same DNA strand as the regulated gene: promoter, enhancer, intron/exons etc. (addition of an acetyl - CH3CO group) for example, results in decreased condensation of the DNA and increased transcription of genes in that region.

Trans: includes all the rest regulatory elements such as transcription activators and repressors.

Regulation of mRNA stability & degradation

The stability of mRNAs is very variable

A deadenylase enzyme acts to shorten the poly-A tail in the cytoplasm

Decapping enzymes - uncapped mRNA is rapidly degraded by exonucleases

3’ binding of miRNA mediated degradation

RNAi mediated degradation

Common methods in genetics

DNA gel electrophoresis

PCR: Polymerase Chain Reaction

Conventional DNA sequencing & High-throughput sequencing

Micro-array hybridization

RFLP: Restriction Enzyme Fragment Polymorphism:

Blotting techniques

Southern Blot

Used to detect DNA

Northern Blot

Used to detect RNA

Western blot

Used to detect protein

TYPES OF BLOTTING TECHNIQUES

Restriction Endonuleases

Restriction endonucleases are enzymes that cleave DNA

molecules at specific nucleotide sequences depending on

the particular enzyme used. Enzyme recognition sites

are usually 4 to 6 base pairs in length.

The recognition sequences are randomly distributed

through the DNA.

SPECIFIC PALINDROMIC SITES

CLONING PROCESS Gene of interest is

cut with specific RE

Host plasmid is cut with the same RE

Gene annealed with the plasmid and ligated with ligase

New plasmid inserted into bacterium (transformation)

STEP 3: LIGATION OF DNA SAMPLE AND PLASMID DNA

Cloning a segment o f DNA into a plasmid vect or

• bacteria are “transformed” with the recombinant plasmid• colonies that grow in tetracycline, but not in ampicillin are isolated

PstIHuman DNA cut with PstI

P

P

pBR322 ampR, tetR

pBR322 (human clone) tetR

P

P

ampR tetR

pBR322 DNA cut with PstIinactivating the ampR gene

tetR

tetR

combineand

ligate

STEP 5: GROWTH ON AGAR PLATES

DNA-Polymerase + Nucleotides

Primers

Denaturation 95°C

Annealing 50-60°C

Extension 68°C

Denaturation, annealing

Extension

x30

Steps in PCR

Microarray Technology: Detection of differentially-expressed genes

Sample Questions

1. DNA molecules can be cut into sections by using:

a. ATP

b. Gel electrophoresis

c. Restriction endonucleases

d. Plasmids

e. A probe

Sample Questions

1. DNA molecules can be cut into sections by using:

a. ATP

b. Gel electrophoresis

c. Restriction endonucleases

d. Plasmids

e. A probe

Good Luck !!!