Exam 4

What question was Avery’s Experiment attempting to answer?

What type of molecule contains the genetic information of the cell? Protein or DNA?

What method did Avery use to determine the genetic information of the cell?

Use the principle of transformation. The S gene was incorporated into the R cell, transforming it to an S cell. Happened after S cell was heat killed and R cell was added. Added proteinase, ribonuclease, and deoxyribonuclease to different cell, and the only time the S gene was destroyed so the R cell did not transform into the S cell was with deoxyribonuclease.

What is transformation?

Living bacterial cell uptakes genetic information from dead bacterial cell

What is the conclusion of Avery’s experiment?

In both eukaryotes and prokaryotes, DNA is the genetic material.

What are Chargaff’s Rules?

A=T and G=C, percentages vary between species, is the same in different cells within an individual organism, and does not change with age, nutrition, and environment.

What did Rosalind Franklin discover?

X-ray diffraction to study DNA structure (hydrated), proved helical structure with structural repeats , and is double stranded.

What did Watson and Crick discover?

Double stranded DNA, helical structure, strands interact via H-bonds of base pairs

Which bases pair with 2 H bonds?

Adenine and Thymine

Which bases pair with 3 H bonds?

Cytosine and Guanine

What are the repeating units of a DNA strand?

Deoxyribonucleoside 5’ mono-phosphates (dNMP)

How are nucleotides connected within a strand?

Phosphodiester bonds

What is the orientation of DNA strands?

Antiparallel

How are DNA sequences read?

5’ - 3’

Why don’t we want covalent interaction between strands?

Strands would not easily separate, limited replication, transcription therefore translation

What conformation of DNA is found most commonly in living cells?

B-DNA

What are characteristics of B-DNA?

Right-handed DNA, mostly flat (propeller twist), base pairs exposed in the major and minor grooves, base pairs lying flat and perpendicular to the axis

How/where are nitrogenous bases exposed?

Major and minor grooves. Sequence specific info comes from nitrogenous bases

Sequence specific info comes from

nitrogenous bases

What is A-DNA?

Conformation of dehydrated DNA

What conformation is a good model of dsRNA and RNA-DNA hybrid molecules?

A-DNA

What is DNA?

Least common form in cellular DNA. Left handed helix

Where is Z-DNA found?

Regions with high G-C content

Denaturing

Separating two DNA strands

Anneal

Complementary single strands of DNA. Does not require enzyme

What does RNA have that DNA does not?

The sugar has a hydroxyl-group at C2 whereas DNA has an H instead

What base replaces T in RNA?

Uracil (U)

Is RNA still read 5’-3’?

Yes

dsRNA and DNA-RNA hybrid molecules resemble ____ conformation.

A-DNA

RNA can base pair with complementary regions of ____.

RNA and DNA

RNA strand orientation

Antiparallel

___ - ___ is allowed when RNA base pairs with itself or another RNA molecule.

G-U

Internal base pairing within an RNA strand yields complex ____.

secondary and tertiary structures

RNA primary structure

Sequence, does not have to be just a straight structure. Stem, loop

RNA secondary structure

Complementary sequence within sequence, base pair with each other in anti-parallel fashion. Hairpin loop, dsRNA, ssRNA, internal loops, stem loops

RNA tertiary structuer

Secondary structure interacts with each other, like tRNA

What is a gene?

Unit of heredity. A gene includes encoding a function RNA or protein and the regulatory elements controlling its expression.

Location of genes is in the ____ position in the entire genome.

same. The positions are all fixed.

___ chromosomes make up genome.

All

What is a chromosome?

Contains a DNA molecule that encodes genes. Eukaryotes have protein bound to chromosomal DNA that package and regulate

What about genes is characteristic of our species?

The position

Diploid cell

2 copies of each chromosome

What pair are the sex-chromosomes?

23rd pair

Humans have __ base pairs, _____chromosomes, ___and genes than e. coli.

More, more, and more

DNA polymerase

Adds one dNMP to the 3’ end of a growing DNA chain. Requires template and primer

RNA polymerase

Adds one NMP to the 3’ end of a growing RNA chain. Requires a DNA template. Will form phosphodiester bond between 2 NTPs to being RNA synthesis. The 5’ end will contain 3 P groups.

Reverse transcriptase

Adds one dNMP to the 3’ end of a growing DNA chain. Requires RNA template and a primer

What is an endonuclease?

breaks a phosphiediester bond within a polynucleotide chain

What is an excinuclease?

Breaks two phosphosphodiester bonds within a single polynucleotide chain

What is an exonuclease?

Removes nucleotides from one end of a polynucleotide chain. Specific for digesting a polynucleotide from either the 5’ or the 3’ end. Break 1 phosphodiester bond at a time from the end

What is a DNA ligase?

Link two existing DNA chains together end-to-end by catalyzing formation of phosphodiester bond. Joined ends result in a continuous 5’-3’ DNA strand

What are the substrates of polynucleotide synthesis?

nucleoside triphosphates

What determines what nucleotides are added?

Template strand

DNA synthesis reaction

(dNMP)n+dNTP→(dNMP)n+1 +PPi, DNA Polymerase, Can only add to existing strands

RNA synthesis

(NMP)n+NTP→(NMP)n+1 +PPi, RNA polymerase can initiate de novo synthesis

What is a nick?

Phosphodiester breakage in only one strand

What is a double-strand break?

Phosphodiester breakage in both strands. Can be a blunt-cut (same position) or staggered (2 different places, leaves sticky ends)

What is a palindromic sequence?

Reads same forwards and backwards

What are restriction enzymes?

Sequence-specific endonucleases

What does DNA ligase need to reseal DNA?

2 pre-existing strands, must follow 5’-3’

Where does AMP from for DNA ligase?

ATP for eukaryotes, and NAD+ for bacteria

How does DNA ligase seal?

DNA ligase has amino group, binds to phosphophate group of AMP, have enzyme-AMP attack the phosphophate of the the nick, then have nucleophilic attack of hydroxyl on phosphate, then release AMP and ligase

Why do we add AMP to the nick?

Makes a good L. G to allow nucleophilic attack of hydroxyl

How to make sticky ends?

Use ligase to make continuous strand

How can we use restriction enzymes in medicine?

Make tons of bacteria, produce lots of proteins (insulin) to give to diabetics

mRNA

Encodes the primary AA sequence for protein, serves as the template for translation by ribosome

tRNA

Carries AA into catalytic site of a ribosome. Base pairs to mRNA to ensure selection of the correct amino acid for incorporation into a nascent polypeptide chain

rRNA

structural components of a ribsome, the enzyme that catalyzes translation

What is the reverse complement?

DNA template strand of coding strand and primary transcript.

and have the same bases except for T being replaced with U.

Coding strand RNA primary transcript.

What do prokaryotes do with the primary transcript?

No modification, can happen simultaneously with transcription and translation

The coding and template strands are defined for __ individual gene, for both eukaryotes and prokaryotes.

every

Cis-acting elements

closely tied to the gene. Typically DNA sequence. Regulatory sequence, fixed location in genome

Trans-acting elements

Diffusible, so they can function at multiple sites in a genome. Usually DNA binding proteins such as transcription factors.

elements bind to elements to activate or deactivate transcription.

Trans, cis

When does RNA polymerase start and stop transcription?

Starts at promoter and does not stop until terminator sequence

What is +1?

Transcription start site

What is an open reading frame?

Sequence of bases that encodes the primary sequence of a protein, aka the coding sequence

What are operons?

Coordinately regulated gene clusters in bacteria.

What is a polycistronic mRNA?

Use of one promoter and terminator yields this, with multiple ORFs each encoding a different protein

What is a promoter?

Cis-acting element, RNA polymerase binds here

What is the bacterial consensus promoter sequence?

TTGACA-N16-18 - TATAAT-N5-9CAT

Where is the bacterial promoter sequence located?

Between -35 and -10

What is a spacer?

Distance is what matters, not the nucleotides

What is the major determinant of gene expression in bacteria?

Rate of transcription initiation

What are strong promoters?

High sequence identity with the promoter consensus sequence. Higher strength of promoter means higher affinity for RNA pol, which means more interactions with DNA, so more protein-DNA interactions

What are weak promoters?

Have several base differences, deviates, less interactions, so lower affinity for RNA pol to bind

What are the two forms of RNA polymerase in e. coli?

Holoenzyme and core enzyme

What is a holoenzyme?

Central enzyme and accessory proteins, has the sigma subunit. Version of RNA polymerase that scans DNA for promoters to see where it can bind, leave sigma behind to then become core enzyme.

What is a core enzyme?

Does bulk of elongation, has 5 subunits. Carries out transcription, does not initiate transcription

What do the alpha subunits in the core enzyme do?

Two identical subunits; important for overall assembly, and interact with transcription factors

What does the omega subunit do?

Stability

What do the ß and ß’ subunits do?

Comes together to make catalytic core

Why is the sigma factor so important in the holoenzyme of RNA polymerase?

The only subunit that can bind to promoter on DNA.

Why can RNA polymerase initiate synthesis de novo?

Does not require primers

What are the differences between RNA polymerase and DNA polymerase mechanisms?

The substrate, and RNA has no proof-reading

What is replication?

Duplication of genomic DNA. Daughter cell inherits a complete complement of the genetic information

What is conservative replication?

Yields an original intact DNA molecule and entirely newly synthesized DNA

What is Dispersive replication?

hybrids/patchwork of parental and new DNA

What is semi-conservative replication?

Yields two DNA molecules, each with one parental and new DNA strands

What is the Meselson and Stahl Experiment?

Attempted to find out how DNA replicated. Used isotopes of nitrogen, e. coli, and a CsCl gradient. Start off by making heavy e.coli, then grow in light nitrogen medium . Then have second replication round