Biology - Introduction to Molecular Genetics

DNA → RNA →Proteins

Central Dogma of Molecular Genetics

What enzyme is used to go from RNA → DNA

Reverse Transcriptase

Nucleoside

sugar (ribose/deoxyribose) + nitrogenous base; no phosphate

Nucleotide

sugar (ribose/deoxyribose) + nitrogenous base + phosphate

Ribonucleic Acid (RNA)

• -OH is present at 2’ and 3’

Deoxyribonucleic Acid (RNA)

• -OH ONLY at 3’

Which Nucleotide is a Purine

• A and G

• PURe As Gold

Which Nucleotides are Pyrimidines?

• T, U, and C

• CUT the PYe

A pairs with

• T in DNA

• U in RNA

G pairs with

C

How many hydrogen bonds between C and G

3

How many hydrogen bonds between A and T (or U)

2

Nucleosome

Structures composed of DNA wrapped around a histone proteins

Chromatin

DNA packing patterns within a chromosome due to the organization of nucleosomes

Euchromatin

• loosely packed

• DNA is easily accessible for transcription

Heterochromatin

• Tightly packed

• DNA is most inaccessible for transcription

Acetylation

• Addition of negatively charged acetyl group to a histone

• Loosens nucleosome packing

Deacylation

• Removes acetyl group; makes overall charge more positive

• Tightens nucleosome making DNA less accessible for transcription

Methylation

• Can tighten or loosen DNA

• Usually keeps inactivated genes inactivated

Steps of DNA replication

• Initiation

• Elongation

• Replication

Initiation

• Relies on creation of the origin of replication

• A special enzyme will recognize the origin of replication and begin replication

What is an Origin of Replication?; How is it formed?

• The beginning point for DNA replication

• Has a high density of AT bonds; AT only has 2 hydrogen bonds meaning this area has a weaker bond making it easier to pull apart

• Circular DNA has 1; Linear DNA has multiple

DNA in Prokaryotes

• Circular

• One origin of replication

DNA in Eukaryotes

• Linear DNA

• Multiple origins of replication

Initiation Phase Replication Enzymes

• Helicase: unzips DNA by breaking H bonds; Creates replication fork; causes supercoiling

• Topoisomerase: relieves built up tension from supercoiling by nicking the DNA

Helicase

• unzips DNA by breaking H bonds

• Creates replication fork

• causes supercoiling

Topoisomerase

• relieves built up tension from supercoiling

• Periodically cuts one strand to allow DNA to unwind then reattach

DNA Elongation Enzymes

• Single Stranded Binding Proteins (Not an Enzyme)

• DNA polymerase

• Primase

SSBs

Prevents DNA strands from reattaching

DNA polymerase

• Elongates new DNA strands by adding nucleotides

• Needs a free 3’ hydroxyl group

• Adds nucleotides from 5’ to 3’

• Replaces RNA primers (from primase) with DNA

Primase

• places an RNA primer complementary to the DNA strand

• Provides free 3’OH for DNA polymerase; creates starting point for nucleotide addition

Leading strand

• continuously synthesized

• 3’ end faces the opening of the DNA fork

Lagging strand

• Synthesized not continuously

• 3’ end faces away from the opening of the replication fork

Okazaki Fragments

• Caused by the lagging strand

• Short DNA fragments that compose the growing lagging strand

Ligase

• Seals together the okazaki fragments

• Creates coherent complementary strands

Termination

• DNA replication will terminate when the end of the chromosome is reached

• Some nucleotides at the end points go unreplicated and are lost

Telomeres

• Non Coding repetitive DNA segments at the end of the chromosomes

• Buffer against loss of critical genetic information

• Located beyond coding DNA

Telomerase

• Replaces lost nucleotides with new ones maintaining the telomeres

In which direction does DNA polymerase read the template strand?

3’ to 5’ (opposite of how it adds nucleotides)

Messenger RNA (mRNA)

• Protein encoding RNA

• RNA that is part of DNA → RNA → Protein

RNA polymerase

• Enzyme responsible for reading DNA template and generating a new RNA transcript

Template DNA strand

• also called the anti sense and non-coding DNA strand

• strand transcribed by RNA polymerase and is complementary to the RNA transcript

Coding/Sense DNA strand

• The strand not being read by the RNA polymerase

• Identical to new RNA transcript except T is U

promoter sequence

where RNA polymerase binds to

Translation and Transcription in Eukaryotes

• occur in different location

• Transcription happens in nucleus

• Translation happens in the cytoplasm

Translation and Transcription in Prokaryotes

• occur in the same location

• Transcription and Translation happen simultaneously in the cytoplasm

Initiation of prokaryotic transcription

RNA polymerase opens transcription bubble

Operon

• Serveal genes packaged together and transcribed all at once

• Controlled by one promoter

• Operator region closely associated with promoter

Operon regulation

• repressors bind to the operator region

• promoter bind to the promoter region

lac-operon

• Inactive without stimulation;must be induced to become active

• Genes involved: lacZ lacY and lacA

• Encodes for different proteins which are all involved in lactose metabolism

• Turns on when glucose is not available as an energy source

lac repressor protein

• Constitutively expressed (always produced under normal conditions)

• When lactose is present it is converted into allolactose

• Allolactose binds to repressor protein and activates lac-operon

• Allolactose is an inducer

CAP site in prokaryotes

• found upstream from RNA polymerase

• cAMP activates the cap protein

• Low glucose = high cAMP

• cAMP presenses makes transcription go faster

trp-Operon

• responsible for producing tryptophan

• Default active unless repressed

• Produces tryptophan synthetase which is responsible for Trp production

• Repressed by product Tryptophan

• Low tryptophan = allows for transcription

• High tryptophan = inhibits RNA polymerase form transcription

Transcription factors (Eukaryotes)

• DNA binding proteins that influence transcription

• May help RNA polymerase bind to promoter regions of DNA

• Includes activators and repressors

Promoter Sequences (Eukaryotic Transcription)

• Site of RNA pol binding

• TATA box: common DNA sequence found within promoter; may be recognized and bound by transcription factors

Enhancers (Transcription Factors < Eukaryotic Transcription)

• DNA segments that bind activator proteins

• When activator proteins bind this increase gene transcription

Silencers (Transcription Factors < Eukaryotic Transcription)

• DNA segments that bind repressor proteins

• When repressor proteins bind to there segment there is a decrease in gene transcription

Colocalization (Eukaryotic Transcription)

• DNA bending protein will bend DNA to bring the promoter regions and silencers/Enhancers in close proximity to one another

• Silencers/Enhancers can bind with transcription factors that can then bind with the promoter region to upregulate or downregulate the rate of transcription

Poly-A-Signal ( Termination < Eukaryotic Transcription)

• DNA sequence located within the termination sequence

• Signal for transcription termination and dissociation of RNA polymerase

• Sequence induces polyadenylation (the addition of many A; AAAAAAA); This will cause a weaker bond between the DNA and RNA transcript finally allowing the RNA to finally snap off

Terminator sequence

• includes the Poly-A-tail signal that induces polyadenylation

transcription produces

pre-mRNA

Post-transcriptional modifications

• Functional alterations to the RNA transcript post transcription

• convert pre-mRNA into mRNA

mRNA

• RNA used to encode for proteins in translation

Main types of post-transcriptional modifications

• 5’ capping

• 3’ polyadenylation

• Splicing

5’ capping (Eukaryotic post translational modifications)

• Cap is the 7-methylguanosine cap

• Helps protect the mRNA transcript from enzyme degradation

• Aids in ribosomal attachment to mRNA transcript

3’ poly a tail

• Addition of poly-A-tail

• Helps protect RNA transcription from degradation

• Signals that mRNA is ready for export from the nucleus

Introns

• Segments of DNA that do no code for proteins

• Removed during splicing

Exons

DNA segments that encode for proteins

Spliceosome

Enzyme responsible for splicing during post transcriptional modifications

Alternative splicing

• Pre-mRNA transcription containing many exons can be spliced into many mRNA variations

• Resulting mRNAs will have different exon combinations

• Alternative splicing allows for one pre-mRNA to encode for many proteins

miRNA

• silence expression of mRNA transcripts

• does this by base pairing with part of the mRNA transcript

• A form of Post-transcriptional gene regulation

• Silence mRNA translation

snRNA (Small nuclear RNA)

• combines with proteins to make the functional portions of spliceosomes

snRNPs (Small nuclear RiboNucleoProteins)

snRNA and protein complex; spliceosome

Translation

• mRNA → Proteins

Ribosomes

• Enzyme responsible for facilitating translation of mRNA into proteins

• catalyzes the peptide bond formation of the growing amino acid chain

Eukaryotic Ribosomal Subunits

• 60s and 40s

• Forms the 80s subunit

Prokaryotic subunit

• 30s and 50s subunits

• forms 70s ribosome

Codons

• segment of an mRNA transcript that encode for 1 amino acid of for translations termination

• made up of 3 RNA bases

Codon degeneracy

• Multiple codons encode for the same amino acid

• 64 possible codons; only 20 amino acids

How many amino acids are there?

20

Start codon

• AUG (Methionine)

Stop codons

• UAA, UAG, UGA

• Do not encode for any proteins

• Signal for the end of translation

Open reading frame

• Stretch of mRNA transcript in between the start and stop codon

• Region of ribosome encoding

• codons found in this frame will be part of translation to become proteins

tRNA (transfer RNA)

• tRNA brings amino acid to ribosome to be associated to mRNA

• Links up amino acids

• anticodon reads mRNA transcript; houses 3 bases that complements mRNA

A binding site

• Where the tRNA first enters the ribosome

• Where amino acid binds to the growing peptide chain

P binding site

• houses the aminoacyl-tRNA that holds the growing polypeptide

E binding site

• Exit Site

• site where tRNA is is released from the ribosome; leaving the amino acid

Translocation

• name for the movement of tRNA molecules through the A, P, and E binding sites

• peptide chain in P site is transferred over to A site (where it will bond with the amino acid in the A site); the A site will now hold the polypeptide chain

Translation Termination

• when a stop codon in reached

• a release factor (with no new amino acid) will bind instead of a tRNA

• Polypeptides chain is released from the last tRNA into the cytoplasm

• The ribosome will dissociate into its 2 subunits

Protein folding (post translation)

• structure of any protein is directly related to its function

• chaperone protein assisits with folding

Chaperonin proteins

• assist some newly synthesized polypeptide chains in folding to their proper shape

• Ensures proper transition between primary protein structure into secondary protein structure

• present in eukaryotic cells and prokaryotic cells

Base Substitutions (DNA Mutations)

when one nucleotide is replace by another

Silent mutations (DNA Mutations)

• Single nucleotide change does not change the encoded amino acid

• Relies of codon degeneracy (when codons encode for the same amino acid)

Missense (DNA Mutations)

• single nucleotide change that changes the encoded amino acid

Nonesense mutation (DNA Mutation)

Conservative missense mutations

• when the missense mutation does not change the polarity (polar/ non-polar) of the molecule

Frameshift mutations (DNA Mutations)

• Insertion: Nucleotide addition from the DNA sequence

• Deletions: Nucleotide removal from the DNA Sequence

Null mutations (DNA Mutations)

• type of loss of function mutation where a null allele is produced ( a non-functional allele)

Chromosomal mutations

• Affect an entire chromosome rather than just one nucleotide or a small series nucleotides

• Include:

  • Deletion

  • Duplication

  • Inversion

  • Translocation

Deletion (Chromosomal Mutation)

• Portion of a chromosome is deleted

• Chromosomal arm shortening