Exam 2

Genome

All genetic information that defines a species, chromosomes and plasmids

Plasmid

Single, circular, small DNA that contains genes that are not essential for survival

What is DNA in a eukaryote like?

Long, linear chromosomes in the nucleus and circular DNA in the mitochondria

What is DNA in a prokaryote like?

Supercoiled and compacted, wrapped around histone-like proteins to allow DNA to fit in the nucleoid

What are the steps of prokaryote DNA replication?

1. Topoisomerase 1 cleaves one strand of DNA at the origin of replication to create replication bubble. This stops strains from tangling while being replicated.

2. The replication fork forms.

3. Primase adds RNA primer

4. DNA polymerase 3 synthesizes the new DNA strand from this primer

5. DNA polymerase 1 replaces RNA primers with DNA

6. Ligase seals gaps

What does helicase do?

Breaks H-bonds between DNA strands to unzip the DNA

What do single stranded binding proteins do?

They prevent DNA from closing while it is being replicated

What direction is DNA synthesized?

The template strand is read from 3’ to 5’, but the new strand will be synthesized by adding onto the 3’ end

What is the difference between the lagging and leading strand?

The lagging strand needs more RNA primers added and will have Okazaki fragments

What does DNA gyrase do?

Prevents supercoiling

What does primase do?

Adds RNA primers for DNA polymerase 3 to synthesize off of

What does DNA polymerase 3 do?

Synthesizes DNA by adding nucleotides to the 3’ end

What is a coding strand?

Identical to the strand being made

What is the template strand?

Complimentary to the strand being synthesized, the template that is used for replication

Nucleotide activation

Nucleotides must be activated through phosphorylation to be polymerized

RNA polymerase

Polymerizes RNA from a template strand, made of sigma and core subunits

Sigma subunit

Directs RNA polymerase to the genome at the promoter sequence, activates a set of genes

Promoter

A DNA sequence that indicates where to start transcription, -35 to -10 bp up from gene, determines the promoter used

Core subunit

Made of 2 alpha, beta, and beta’ subunits, catalyzes the polymerization of RNA

Consensus sequence

Average of DNA sequence that do one job

Strong consensus sequence

Very close to the sequence, stronger initiation, more mRNA produced, for proteins needed in large amounts

What are the steps of RNA synthesis?

Initiation, elongation, termination

What are the types of termination?

Rho-dependent and Rho-independent

Rho-independent termination

When a termination sequence is transcribed the RNA folds into a hairpin loop and gets in the way of RNA polymerase, no ATP used

Rho-dependent termination

Rho protein binds to mRNA at the RUT site to knock the RNA polymerase off, ATP is used

RUT sites

C every 12 bp, can be blocked by ribosomes, cause Rho proteins to bind to them to end transcription

What is the RNA pair for C?

G

What is the RNA pair for A?

U/T

How many proteins are in a cell at once?

Up to 1000 kinds with different numbers of copies

Copy number

How many proteins of a specific gene there are

tRNA

Transfer RNA, matches amino acids with specific nucleotide sequences, activate amino acids

When is a tRNA activated/ chagred?

When it’s connected to an amino acid

When is a tRNA inactive/uncharged?

When not connected to an amino acid

Ribosome

An enzyme that polymerizes activated amino acids, ribonucleic acid, made of 30s and 50s subunits

What is the 30s subunit made of?

16s rRNA and 21 polypeptide chains

Ribonucleic acid

Made of RNA and proteins

What is the 50s subunits made of?

5s rRNA, 23S rRNA, 34 polypeptide chains

Initiation of translation

1. 3’ of 16s of the 30s rRNA base pairs with the complementary site of the mRNA

2. The 50s subunit sandwiches the mRNA

3. The translation start site is reached 6-9 base pairs downstream

Elongation of translation

1. Activated tRNA are delivered to ribosome by elongation factor TU

2. Peptide bonds form between amino acids

3. Elongation factor G slides ribosome 3 base pairs downstream on the mRNA

4. Repeat

Ribosomal binding site

RBS, same as shine dalgarno (SD) site, where ribosome binds to mRNA to start translation

Termination of translation

1. The stop codon is reached

2. Release factors recognize stop codon and arrest protein synthesis

What direction are proteins synthesized in?

From C to N

What catalyzes peptide bond formation in translation?

23S rRNA in the ribosome

Release factors

RF proteins, bind to stop codon and release ribosome from mRNA. Ribosome then dissassembles

What is the first amino acid?

fMET

Tetracycline

Blocks amino acyl tRNA entry

Chloramphenicol

Blocks peptide bond formation

Erythromycin

Blocks ribosome translocation

Gene

Aka cistron, segment of DNA that codes for a functional product

Operon

Multiple genes expressed by a promoter, one set of regulatory elements needed, 1 mRNA strand is made, genes have their own SDS and translational stop and start, keeps related genes together

What are the ways to regulate translation?

Consensus sequence, SD site strength, alternative sigma factors, DNA binding proteins, riboswitches/ attenuators, regulatory small RNA

Alternate sigma factors

Different sigma factors recognize different promoter sequences, coordinates genes with similar functions, can cause a cascade

Antisigma factors

Attach to a sigma factor to keep them inactive until needed

DNA binding proteins

Bind to a sequence to increase or decrease gene expression

Activator

Activates downstream genes by stabilizing RNA polymerase interactions with the promoter, binds to DNA to increase gene expression

Repressor

Turns off expression when bound to DNA to block RNA polymerase from promoter region/ changing the promoters to change the shape to prevent binding, binds to operator

How does the lac operon work when glucose is absent?

1. CAMP is synthesized by ADC using ATP when glucose is not present present

2. CAMP binds to CAP and activates it

3. CAP activates the lac operon and the lac proteins are transcribed

How does the lac operon work when glucose is present?

1. Glucose deactivates ADC and cAMP cannot be made

2. CAP can’t be activated

3. The lac operon is not activated and no proteins are transcribed

How can repressors prevent transcription?

Steric hinderance, DNA looping, antiactivation, protein-RNAP interaction

Steric hinderance

The repressor has a higher affinity for the promoter than the RNA polymerase. By binding the repressor inhibits transcription

DNA looping

2 repressor proteins bind at distance sites and interact to cause the DNA to bend and stop the RNA polymerase from being able to access the promoter

Antiactivation

Repressor binds to the promoter and prevents the activator protein from binding, which inhibits transcription

Protein-rRNA interaction

Both RNA polymerase and the repressor are bound to the promoter region, but the repressor blocks ribosome assembly

Positive regulation

Sensing a change in the environment and changing gene expression to turn on transcription of a gene

Negative regulation

Sensing a change in the environment and changing gene expression to turn off transcription of a gene

One component system

The same molecule sense a change and binds to DNA to change gene expression, 1st kind of system to evolve

Two component system

A kinase senses conditions outside of the cell and passes the message to a response regulator in the form of a phosphate group. The response regulator changes gene expression. Causes a cascade. The signal can change but the kinase and regulator will be the same.

Chromatic acclimation

Protein production of light absorption pigments adjusts to match light color

What do phosphates do to proteins?

Changes their structure

Kinase

An enzyme that has a phosphate

Systems biology

The study of how complex biological interactions lead to cellular functions

Riboswitches

5’ end of mRNA binds to a molecule and causes the mRNA to fold into a structure that changes gene regulation, can be activators or deactivators. Use less energy because a DNA binding protein/sigma factor doesn’t have to be produced

What do riboswitches respond to?

Vitamins, some amino acids, glucosamine-6-phosphate, and cyclic di-GMP

Flavin

A precursor molecule

How do riboswitches act when there are low concentrations of flavin?

The mRNA spontaneously folds and trasncription occurs

How do riboswitches work when there are high concentrations of flavin?

mRNA spontaneously folds, a ligand binds, and there is early termination

Small regulatory RNAs

sRNA, untranslated regions of DNA that have a regulatory function

Antisense RNA

Bind to complimentary sequences and affect translation. Can up or down regulate genes

How can sRNAs downregulate a gene?

Block ribosomal binding site and destabilize RNA by recruiting a RNase

How can sRNA upregulate a gene?

Can cause mRNA to change shape and expose a ribosomal binding site or stabilize RNA by preventing RNase from cleaving it

Induceable

Gene is turned on when a stimulus is added

Repressible

Gene is turned off when a stimulus is added

Mutation

Heritable change in DNA, can be good/bad/neutral

What causes more random mutations?

A weaker proof reading when replicating DNA

Recombination

Physical exchange of DNA

What are the sources of mutation?

DNA polymerase adding the wrong base, radiation, and chemical mutagens

How can radiation cause a mutation?

Damage DNA or cause T dimers that cause bumps in DNA that change transcription

How do chemical mutations cause mutations?

Base analogs are included instead of bases, nucleotide altering chemicals change bases, and intercalating chemicals

Intercalating

Chemicals that insert themselves between stacked base pairs. Can disrupt translation, cause a change to DNA structure, and cause frameshift mutation

When are the effects of mutations minimal?

In non-coding regions, non-translated mRNA, or the 3rd base

Wobble

Changing the 3rd nucleotide of a codon has no effect on the amino acid coded for because all 4 bases code for the same amino acid

Silent mutation

No change to the protein

When are there more consequences to a mutation?

A single base change to a consensus sequence, missense mutations, and when amino acids are changed

What genes/regions of DNA have a consensus sequence?

Promoter, ribosomal binding sequence, regulatory regions

Missense mutation

A mutation that causes the protein to change

Nonsense mutation

A mutation turns a codon into a stop codon and causes early termination

How do insertion/deletion mutations effect the protein?

The reading frame shifts and this changes protein structure

Open reading frame

Checks reading frame forwards and backwards, looks for stop and start codons

Recombination

Horizontal gene transfer, how