Unit 6: Gene Expression and Regulation

Ch. 16, 17, 18, 19, 20, 21, 27.1-2

Nucleotides

comprised of a 5-carbon sugar, a nitrogenous base, and a phosphate group.

Purines

(A,G) have two rings in their nitrogenous bases.

Pyrimidines

(C,T,U) have one ring.

Prokaryotic DNA

1 circular chromosome (millions of base pairs), and usually at least one small, extrachromosomal plasmid (thousands of base pairs)

Eukaryotic DNA

multiple linear chromosomes (e.g. humans have 46 - ~3 billion base pairs)

Viral DNA

comprised of DNA or RNA, single or double stranded (thousands of base pairs).

Exons

sections of DNA pulled out in order to be expressed

Introns

sections of DNA that are left behind

Semi conservative model

• each strand of old molecule serves as a template for the synthesis of a new strand

Replisome

• All of the enzymes involved in replication that function together.

Helicase

opens the helix by breaking the (weak) hydrogen bonds between nucleotides

Ligase

forms covalent bonds between adjacent nucleotides in one strand of DNA. Needed to join Okazaki fragments together.

Topoisomerase

Rotates the helix ahead of the replication fork to reduce stress.

DNA Polymerase III

• Responsible for the addition of free nucleotides to a new growing strand of DNA.

• Can only add nucleotides in the 5’ → 3’ direction of the growing strand.

• Makes mistakes but does proofreading to fix them.

• Synthesizes new strands by adding to RNA primer or pre-existing DNA strand only.

DNA Polymerase I

• Responsible for removing RNA nucleotides of primer from 5’ end and replacing them with DNA nucleotides.

• can only add nucleotides to the 3’ end of a molecule (moving in the 5’ → 3’ direction of the growing strand).

• Note: Nucleotides must start building from a DNA primer, polymerase removes the primer and begins DNA replication

• Can only attach to a primer or base already present. Primer, made of RNA, acts like a flag to indicate where DNA synthesis begins. Finally, the U’s in the RNA are taken out by DNA polymerase (removing primer) so that the DNA is official.

Primase

Synthesizes RNA primer at 5’ end of leading strand and 5’ end of each Okazaki fragment

Leading Strand

• synthesized continuously in 1 piece

• synthesized as DNA polymerase moves along the template as the replication fork progresses

Lagging Strand

• synthesized discontinuously in multiple fragments, connected by ligase

• series of short segments, AKA Okazaki fragments,

Topoisomerase

Rotates the helix ahead of the translation fork to reduce stress

Nuclease

• During DNA replication, DNA polymerase synthesizes the new strand in the 5' to 3' direction. If it detects an incorrect nucleotide, it uses its 3' to 5' exonuclease activity to remove the incorrect base before continuing synthesis.

  • TLDR; 3’ to 5’ nuclease CUTS OUT ERRORS

Cuts out DNA that is damaged or has errors

Telomeres

• Adds a little bit more protective sequence to start RNA primer earlier, covers the part that is supposed to be removed. Will eventually lose it, but protects it for the time being.

regions at end of chromosomes that contains repetition of bases that do not contain genes. act as protectors of the bases that DO contain genes.

Telomerase

Catalyzes the lengthening of telomeres in eukaryotic germ cells to compensate for their shortening in DNA replication.

Poly Adenylation tail

a chain of Adenine residues is added to the 3’ end of the transcript. Aids in transport of the mRNA from the nucleus and increases the longevity of the transcript.

not coding for anything, just adding buffer onto tail

5’ guanine capping

a modified nucleotide (GTP cap) is added to the 5’ end of the transcript. Similar in function to the poly A tail; prevents degradation but also facilitates export from the nucleus

Exon slicing

Many segments of the transcript (“introns”) are removed, and the remaining segments (“exons”) are spliced together to produce a mature transcript.

Splicesosomes

are made of proteins and small RNAs. The small RNAs within the spliceosome recognize the intron–exon boundary, catalyze the excision of introns from the pre-mRNA, and join together the exons.

mRNA

rRNA

Structural components of ribosome subunits.

Regulatory RNA

Control gene expression by blocking transcription.

Transcription

Converts DNA sequence information into RNA sequence information.

RNA Polymerase

Enzyme that catalyzes the 5’ → 3’ synthesis of an RNA strand from a single DNA strand. Doesn’t require a primer to begin synthesis (unlike DNA polymerase).

Translation

Converts mRNA sequence information in to polypeptide sequences.

Occurs at the ribosome.

Genetic Code

The genetic code is interpreted as a series of 3-nucleotide codons. 64 possible codons, which code for all 20 amino acids, along with 1 START codon and 3 STOP codons.

“Redundant, Unambiguous, Punctuated”

Redundant – many codons code for a single amino acid.

Unambiguous – one codon does not code for multiple amino acids.

Punctuated – no overlapping of codons in sequence.

Initiation

The mRNA interacts with the ribosome to begin translation at the START (AUG) codon closest to the 5’ end of the mRNA.

Elongation

Subsequent amino acids are brought to the ribosome as specified by subsequent, adjacent codons.

Each amino acid is transferred to a growing polypeptide chain.

Termination

This process continues until the first STOP codon is reached, which triggers the release of the polypeptide and the disassembly of the ribosome.

Substitution

• Change one base to another. Can affect the structure of a protein by changing one amino acid, or changing the placement of a stop codon.

  “point mutation”: 1 point in the genome is being affected.

Insertion/Deletion of bases

• “Frame shift mutations”:  Can affect the structure of a protein by changing the amino acid sequence of all subsequent amino acids following the ______, or changing the placement of a stop codon. Called a “frame shift” mutation because it affects the “reading frame” of the ribosome.

• Every amino acid that follows is different, COMPLETELY changes all the amino acids that are translated

Promoter

• sequenced in front of a transcribed gene recognized by RNA polymerase and transcription factors.

• The presence of transcription factors on the ______ make transcription possible.

regulatory sequence

Enhancers

• sequences further “upstream” of a gene that increase the rate of transcription (“upregulation”).

  • increase transcription by bringing two strands of DNA closer together

regulatory sequence

Operons

Groups of metabolically related genes with a single promoter // Common in prokaryotes

Repressor Protein

a regulatory protein that allows/blocks transcription by physically associating/disassociating with a region of the promoter called the “operator”

Inducible Operons

usually “off” but can be induced “on” in the presence of inducer molecule

Repressible Operon

usually “on” can be induced “off” in the presence of corepressor molecule

RNA Interference (RNAi)

the blocking of transcription by small interfering RNA or micro RNA molecules, which bind to specific transcripts and keep the ribosome from being able to translate them or degrade the mRNA.

• RNA will recognize viral DNA and bind to prevent the ribosome from transcribing

Protein targeting

signal sequences on polypeptides will determine if the polypeptide will be made in the cytoplasm or if the ribosome will associate with the ER.

Ubiquinone/Proteasome degradation

Tagging of a protein by a ubiquinone molecule will cause the protein to be degraded by a “proteasome” complex.

Proto-oncogenes

becomes oncogenes when mutation causes uncontrolled cell growth

genes that code for proteins responsible for normal cell growth

Oncogenes

cancer causing genes

P53 gene

• Activating P21 gene which halts cell cycle by binding to CDKs until DNA is repaired.

• Activating miRNAs which inhibit cell cycle.

• Turning on genes directly involved in DNA repair.

• Activating apoptosis if DNA is beyond repair.

tumor suppressing gene

Plasmids

Small circular double stranded DNA molecules.

• can be acquired and replicated by prokaryotes

• May carry genes that help prokaryotes survive

Restriction Enzymes

• This enables the isolation of different segments of DNA (making “restriction fragments”), for introduction into plasmids, sequencing, or a variety of other studies. 

Found in bacteria and cuts foreign/viral DNA at specific sequences. The bacterial cell’s own DNA is methylated in a way that prevents attack by its own ______________.

Horizontal Gene Transfer

• very common in modern prokaryotes in ancestral development of all lineages

exchange of genetic material among members of the same generation (can be the same species or from another species)

Transduction

transfer of DNA between bacteria due to mistakes in replication

Conjugation

• 1. A pilus of the donor cell attaches to the recipient, pulling the two cells together like a grappling hook. 

• 2. Formation of a temporary “mating” bridge structure between the two cells which the donor may transfer DNA to recipient.

• 3. F factor exists either as plasmid or segment of DNA within the bacterial chromosome.

(add image later)

Transfer of DNA due to cell to cell contact

Transposition

• Transposons move within a genome using the “cut and paste” mechanism, which removes the element from the original site.

The replication and movement of genetic elements to sites within the same or on a different chromosome.

Retrotransposons

move within a genome using the “copy and paste” mechanism, which always leaves a copy behind—by producing RNA that is transcribed by reverse transcriptase into DNA that is then inserted at a new site.

Genome Evolution

Duplication, transposition, rearrangement, and other mutations of DNA contribute to genetic variation

Internal Signals

• Regulatory gene, control which genes develop into body parts

• Slight mutation will change the structure of body parts (maybe 2 less legs)

  • Slight mutation causing such a big change means it is HIGHLY CONSERVED

EXAMPLE: Hox/Homeotic gene products: control the development of animal body segments (master regulatory genes)

Viruses

Intracellular parasite

• consists of nucleic acid & a protein coat.

  • some eukaryotic viruses also have a lipid envelope

(+)ssRNA: (positive sense)

serves as mRNA, can be directly translated into proteins in host cell

(-)ssRNA: (negative sense)

complementary to the mRNA that it encodes (*anticodons) —cannot be translated into protein directly. It must first be transcribed into a (+)ssRNA that acts as mRNA. Usually carries RNA replicase enzyme with it.

Infection

the virus is able to transfer its genetic information into a host cell. Viruses are specific to particular cell types. This is due to the specificity of the receptors they use to attach to host cells.

Replication (Viral)

the viral genome (and viral polymerases if necessary) utilize host cell materials to manufacture viral proteins and replicate the viral genome.

Self-assembly

new viral particles are spontaneously assembled from their components.

Release

viral particles are released into the environment to infect new cells

Lytic - phages directs production of an enzyme that damages bacterial cell wall, allowing for water to come into cell and rupture (releasing the bacteria)

Lytic Cycle

Bacteriophage injects DNA into host cell, takes over host cell’s machinery, synthesizes new copies of viral DNA/coat proteins. These self-assemble and release when cell lyses (ruptures).

Phages that replicate only via the _______ cycle are known as virulent phages.

A replicative cycle that ends with CELL DEATH.

Lysogenic Cycle

Bacteriophage DNA becomes incorporated into host

cell’s DNA and is replicated along with host cell’s genome.

The viral DNA is known as a prophage.

Replication of the viral genome WITHOUT death of host cell.

Temperate Phages

Phages that replicate using both lytic and lysogenic cycles

Retroviruses

• uses Reverse Transcriptase to transcribe viral RNA into DNA

(EX: HIV) viral genome is reverse transcribed into DNA (ie RNA → DNA) and integrated into the host cell’s genome. The eventual immune system collapse causes AIDS, unless the infection is halted.

Viral Polymerase

• no ability to proofread allows for more genetic variation

  • Think of the flu: must get new vaccination every year because strand changes just slightly

make many more mistakes than cellular polymerases.

Viral Recombination

co-infection of a cell by 2 viral strains, creating a stronger, extremely pathogenic virus

Gel Electrophoresis

Technology that

• Utilizes an electrical field and a gel matrix  

• Separation of DNA molecules based on their size 

• Smaller DNA molecules migrate through the gel faster than larger molecules 

• Used to isolate specific genes from within larger samples of DNA, or to visualize differences in DNA sequences

Restriction Fragment Length Polymorphisms (RFLP)

Differences in restriction fragment lengths of a DNA sample produced by a given restriction enzyme

Analysis: Using differences in restriction fragment length to determine identity, identify traits, etc.

Prokaryotic Genome

• Have a single, circular DNA molecule located in the nucleoid region (not enclosed by a membrane). They may also have plasmids, which are small circular DNA fragments.

• Lack membrane-bound organelles. Cellular processes occur in the cytoplasm or on the plasma membrane.

• Reproduce asexually, transfer DNA through horizontal gene transfer (conjugation, transformation, transduction)

Eukaryotic Genome

• linear chromosomes contained within a membrane-bound nucleus. typically much larger and organized into multiple chromosomes.

• membrane-bound organelles such as the nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus, and lysosomes, allowing for compartmentalized functions

• asexually (mitosis) or sexually (meiosis and fertilization), which promotes genetic diversity

Bacterial Transformation

1. Uptake – Bacterium absorbs free DNA from surroundings.

2. Integration – DNA may integrate into the genome (via recombination) or exist as a plasmid.

3. Expression – New traits (e.g., antibiotic resistance) may be acquired.

• Helps bacteria adapt & evolve.

• Used in biotechnology (e.g., genetic modification).

A form of horizontal gene transfer where bacteria take up foreign DNA from their environment and incorporate it into their genome.

Evo-devo (Evolutionary developmental biology)

study of how small genetic changes can lead to major evolutionary differences in body structure

PCR - Polymerase Chain Reaction

In vitro DNA replication

• quickly produces billions of copies of DNA for further analysis

• USES: a target sequence of DNA, small primers to bracket the sequence, a heat-resistant DNA polymerase, and a thermal cycler.

How is DNA read?

3’ to 5’

How is DNA synthesized?

5’ to 3’

Template Strand

• AKA: minus strand, antisense, noncoding strand

strand that is used as baseline for the RNA polymerase to know what to code for in the coding strand

Bacteriophages

viruses that infect bacteria

Reverse Transcriptase

• violates Central Dogma

• RNA → DNA

a specialized enzyme* which transcribes an RNA template into DNA

*not naturally occuring in human DNA, retroviruses will encode for the production of reverse transcriptase

Provirus

integrated viral DNA (animal virus)

Silent Mutation

same amino acid (because genetic code is redundant)

Nonsense Mutation

stop codon instead of an amino acid

Missense Mutation

changes amino acid

• effect depends on chemistry of the substitution

  • EX: nonpolar amino acid swapped for polar amino acid WILL affect shape of protein due to bonds