Bio 221: Unit 2

Frederick Griffith

Discovers transformation before people know that DNA is genetic material

Griffith’s Strep. S

Pathogenic (kills)

Griffith’s Strep. R

Non pathogenic (no effect)

Effect of mixing dead type S with living type R?

Living, pathogenic type S was found in the dead mice

Griffith’s Conclusion

Genetic information is passed between the cells to change them from type R to type S

Avery, McLeod, and McCarty

Expand on Griffith’s experiment to try and isolate genetic material

Step One of Experiment

DNA, RNA, and proteins are purified and introduced to bacteria. Only the DNA transforms bacteria

Step Two of Experiment

DNA is treated with enzymes that break it down, transformation ceased

Difference between RNA and DNA

RNA has an OH group attached to the 2’ carbon

Watson and Crick

DNA is a double helix formation and “immediately suggests a copying mechanism“

Semi conservative Replication

Parent DNA molecule is split into two strands to serve as template strands for daughter strands, which will contain one old strand and one new strand

Meselson and Stahl’s Experiment

Proves DNA is semi conservative in replication through isotope heavy nitrogen media

Synthesis of DNA

5‘ --→ 3’, built by DNA polymerase

DNA Helicase

Binds to replication fork and separates the DNA strands

DNA Topiosomerase

Relieves super coiling by temporarily breaking phosphodiester bonds

Single-Stranded DNA binding protiens

Binds to base pairs during super coiling to prevent them from re-bonding

Number of DNA polymerases in different cells

Bacteria: 3 Eukaryotic Animals: 5

DNA Polymerase

Sits on template strand and reads base pairs, nucleotides are added to the 3’ end by removing two phosphates

Primase

Will synthesize complimentary RNA to the parent strand

Primer

Short strands of RNA attached to the origin of replication in bacterial replication

DNA polymerase 3

Synthesizes both the leading and lagging strands in prokaryotic DNA

Okazaki Fragments

Discontinuous synthesis of the lagging strand allowing for simultaneous synthesis of the leading strand

DNA polymerase 1

removes RNA primers and replaces them with DNA fragments

Nicks

Missing phosphodiester bonds between primers and Okazaki fragments, nicks are dissolved by DNA Polymerase 1

DNA ligase

forms the last bonds between Okazaki fragments

dnaA proteins

DNA replication in prokaryotes is dependent on dnaA, it regulates its own production

dnaA levels…

will increase as cell volume increases, triggering the recruitment of helicase to the origin of replication

Nucleosome

eukaryotic DNA bound in histones, present every 200 base pairs

S Phase

DNA is replicated during S phase

ORC

Origin Replication Complex, needs to bind to the origin of replication before replication is initiated

Licensing Factors

Must be present before replication can be initiated, can cause Meier Gorlin Syndrome if not present

Central Dogma of Biology

DNA-→ RNA--→ proteins

Transcription

DNA is a gene that serves as a template for RNA production

Transcribed Region

Region of the DNA in a gene that serves as a template strand for RNA production

Promoter

Recruits the transcriptional machinery

Regulatory Sequence

Recruits proteins that regulate transcription

Terminator

Terminates production of the RNA molecule by causing transcription machinery to fall off the template strand

\+1 position

First base pair after promoter region

AUG

Start Codon

UAG/UAA/UGA

Stop Codons

UTR

Untranslated region of the transcript

Enhancers

Additional regulatory sequences to regulate gene expression of transcription

Initiation

First major step in **Transcription**. RNA polymerase is recruited to the gene by sigma factors

Preinitiation Complex

GTP and RNAP

Elongation

Second major stage in Transcription. RNA polymerase moves in the 5’--→ 3’ direction along gene using the template strand as a guide for RNA synthesis

Termination

last step in Transcription. Once RNA polymerase reaches termination sequence, it falls off the coding strand and releases the new RNA molecule

RNAP

Makes pre-mRNA

Capping

The addition of a methyl guanosine cap to the 5‘ end to reduce degradation

Tailing/Polyadenylation

the addition of adenosine to the 3’ end to limit degradation and allows RNA to leave the nucleus

Splicing

Removes introns in two steps. Produces a mature RNA by the end

Splicing step one

breaks phosphodiester linkages between intron and preceding exon, after this step the intron is in lariat form

Splicing step two

breaks linkages between the intron and the following exon

Translation

production of a protein from an mRNA

Coding Regions

determine which amino acids are produced, the three nucleotide sequences(codons) correspond to specific amino acids

Components of Translation Machinery

Ribosomes which build protein using mRNA as a guide and tRNA to read genetic code

Ribosome Subunits in Translation Machinery

prokaryotes: 50S(23S ribosomes) and 30S(16S ribosomes) subunits, eukaryotes: 60S(28S ribosomes) and 40S(18S ribosomes)

Anticodons

base paired to codons in transcripts read un the 3’ to 5’ direction

tRNA Charging

amino acids are attached to the tRNA

tRNA charging step one

amino acids, ATP and aminoacyl-tRNA synthase link

tRNA charging step two

synthase binds to specific tRNA by covalent bond linkages

tRNA step three

amino acids are bound to form a charged tRNA

Translation Initiation

begins with the recruitment of the small subunit to the start codon in the transcript

Shine-Dalgarno sequences

base pair that link with 16S rRNA in the transcript, initiator tRNA brings N-formyl methionine to start codon, 50S subunit is then recruited and attached to the start cite

Kozak Sequence

Proteins that surround the start codon in eukaryotes

Translation Elongation

ribosome binds tRNA together in the A,P, and E cites. peptide bonds are formed by linking amino acids from the P cite

Translation Termination

processes of elongation continues until it reaches a stop codon, release factors bind in the A cite and disassembles the ribosome

Gene Regulation

Occurs during transcription, translation, and post-translation in both both prokaryotes and eukaryotes, eukaryotes can also regulate during RNA modification

Gene Regulation in prokaryotes

achieved by changing sigma factors in RNA polymerase, important in translation initiation

Regulatory Proteins

Can either repress or activate transcription

Effectors

Bind to specific sites of regulatory proteins to turn them on of off (called allosteric sites)

the lac operon

Inducible operon that regulates the digestion of lactose

Inducible Operon

An operon that will produce a single transcript that includes multiple coding regions for multiple proteins

Lac 1 Gene

encodes for the lac repressor that regulates and prevents transcription

Hight lactose levels=

High lac operon levels

Allolactose

Bonds to the lac 1 gene/repressor, making it unable to bind to the lac operon, inducing transcription

CAP

in the lac operon, it serves as a positive regulator using cAMP to initiate translation, CAP is regulated by glucose levels

High glucose levels=

low cAMP levels, low CAP activity, low lac operon expression

trp operon

a repressible operon that either blocks or induces RNA synthesis in the presence of tryptophan

Repressible operon

The presence of a substrate causes expression of the gene to decrease

High tryptophan levels=

trp operon expression is low, trp binds to the repressor which allows the repressor to bind to the RNA and prevent transcription

Steps for turning a gene on in eukaryotes

1. Modify chromatin structure
2. Recruit the transcription machinery

How to modify chromatin structure:

Chromatin must be loosened to make DNA more accessible

How to recruit transcription machinery in eukaryotes:

TATA box and initiator site are promoters called the core promoter regulatory factors while general transcription factors surround the core promoters to form the preinitiation complex

Preinitiation complex(PIC) is regulated by

activator and repressor proteins called transcription factors

Acetyl group

A group which can be removed from lysine to loosen chromatin structure

Histone Code

Groups of chemical modifications

Deacetylation

the tightening of DNA after RNA polymerase does translation

Methylation of Cytosine in DNA

Associated with the closing up of chromatin, causing less gene expression

CpG Island

The methylated cytosines next to the guanines, allows a cell to regulate gene expression near the promoter group

Silent genes

Genes that are methylated, unmethylated genes are expressed

Alternative splicing

Determines how a cell functions, introns can be spliced differently or exons can also be removed

Steps of iron level regulation

1. Tr-Fe binds to the iron
2. TfR is a transferrin receptor on the body of a cell that initiates endocytosis
3. Ferrin will bind to and store iron in the cytoplasm of the cell to regulate iron levels

High iron levels=

High ferritin levels, low transferrin receptor levels

Ferritin regulation happens at what stage specifically?

The scanning stage

Degraders of transferrin:

A-U rich elements

Low iron levels=

IRP is activated to reduce degradation of transferrin

RNA Interference

the binding of small, noncoding RNA to target mRNA to prevent the mRNA from being transcribed

microRNA

produced from endogenous transcripts, will inhibit the transcription of mRNA

siRNA

produced outside the cell, triggers the breakdown of RNA

RNA interference evolved to…

protect organisms from viral infections