Genetics: Exam 3 (Chapters 13-15, 18)

Molecular Genetics

Definition: A branch of biology that studies the structure, function, and regulation of genes at a molecular level.

Application: Allows us to build molecular tools for biotechnology and precision medicine.

Both DNA and RNA

Composed of nucleotides and those nucleotides are joined by phosphodiester bonds.

DNA only / not RNA

Deoxyribose sugar, NO presence of 2’ -OH group, T as one of the bases, usually DOUBLE stranded, DOUBLE helix as secondary structure, STABLE

RNA only / not DNA

Ribose sugar, YES presence of 2’ -OH group, U as a base, usually SINGLE stranded, MANY secondary structures, easily DEGRADED

OH group on ribose is chemically more reactive

RNA is less stable compared to DNA

RNA secondary structure

• Primary structure FOLDS to the secondary structure

  • Via intramolecular base pairing

• PROVIDES FUNCTION

RNA in both prokaryotes and eukaryotes

Big 3: rRNA, mRNA, tRNA

RNA in prokaryotes only (everything else is in eukaryotes only)

CRISPER RNA (crRNA)

RNA located in both the nucleus and cytoplasm

mRNA only (middle, only RNA of big 3), microRNA/miRNA (middle), small interfering RNA/siRNA, piwi-interacting RNA/piRNA, long noncoding RNA/lncRNA

RNAs w/ m, i, l (l looks like i) = middle = both

RNA located in cytoplasm only / not the nucleus

rRNA and tRNA (big 3 except mRNA)

cytoplasm = most of cell = needs more of big 3

RNA in nucleus only, not cytoplasm

small nuclear RNA/snRNA and small nucleolar RNA/snoRNA

n = nucleus

Ribosomal RNA (rRNA)

structural and functional components of the ribosome

in BOTH prokaryotic and eukaryotic cells, and ONLY in the cytoplasm

Messenger RNA (mRNA)

carries genetic code for proteins

in BOTH prokaryotes and eukaryotes, and in BOTH the nucleus and cytoplasm

Transfer RNA (tRNA)

helps incorporate amino acids into polypeptide chain

in BOTH prokaryotes and eukaryotes, and ONLY in the cytoplasm

Small nuclear RNA (snRNA)

processing of pre-mRNA

in eukaryotes only and in the nucleus only

nuclear = genes = mRNA (has genetic code)

Small nucleolar RNA (snoRNA)

processing and assembly of rRNA

in eukaryotes only and in the nucleus only

nucleolar = ribosomes

MicroRNA (miRNA)

inhibits translation of mRNA

in eukaryotes only and in both the nucleus and cytoplasm

mi = mRNA inhibitor

Small interfering RNA (siRNA)

triggers the degradation of other RNA molecules

in eukaryotes only and in both the nucleus and cytoplasm

interfering RNA = degrading RNA

Piwi-interacting RNA (piRNA)

suppresses the transcription of transposable elements in reproductive cells

in eukaryotes only and in both the nucleus and cytoplasm

PP = reproductive cells

CRISPR RNA (crRNA)

assists destruction of foreign DNA

in prokaryotes only

cutting of genes

Long noncoding RNA (lncRNA)

eukaryotic nucleus and cytoplasm, variety of functions

Central dogma

DNA (DNA replication) — transcription —> RNA — translation —> protein

scribe before translating

Both DNA replication and transcription

use DNA as a template and synthesis occurs in 5’ to 3’ direction

DNA replication / not transcription

Copies the whole genome to prepare for cell division.

DNA DNA DNA

• Both strands of DNA involved - DOUBLE stranded

• Main enzyme: DNA polymerase

• YES primer needed (DNA 1st ~ primer)

• Final product: 2 identical double stranded DNA molecules

• T base involved

Transcription / not DNA replication

Converts a specific gene into RNA for protein synthesis.

Going to RNA RNA RNA

• Only one strand of DNA gene is used (template strand) - single strand ~ pseudo RNA

• Main enzyme: RNA polymerase

• NO primer needed

• Final product: a single stranded RNA molecule

• U base involved

Requirements for transcription

1. DNA template strand

2. rNTPs = ribonucleoside triphosphates (AUGC)

3. proteins necessary for starting/continuing the production of RNA, one ex. RNA polymerase

DNA strands in relation to transcription

Nontemplate strand: same as RNA (except has T instead of U)

Template strand: opposite to RNA

Where and how is transcription initiated

Gene structure, specifically the promoter

Promoter

DNA sequence upstream of transcription start site, where transcription apparatus binds

RNA-coding region

Serves as a template for RNA synthesis

• includes the terminator but NOT the promoter

• resulting RNA transcript is the same length as the coding region

Terminator

DNA sequence that causes transcription to stop, usually part of the RNA

Parts of DNA for Transcription

Promoter (not included in RNA), RNA-coding region, and terminator

Steps of Transcription

1. Initiation of RNA synthesis does not require a primer

2. Nucleotides added to 3’ end of RNA

3. DNA unwinds at the front of the transcription bubble (3’ of RNA / 5’ end of DNA)

4. DNA rewinds (3’ end of DNA)

Core enzyme of bacterial RNA polymerase

composed of 5 subunits (2 alpha, beta, beta prime, omega) - synthesizes RNAS

Sigma factor

guides the core enzyme to the specific gene promotors to start transcription

Holoenzyme

form of the enzyme that initially binds to promotor (core RNA polymerase + sigma factor = RNA polymerase holoenzyme)

Transcription Initiation

1. Core RNA polymerase + sigma factor = holoenzyme

2. Holoenzyme binds to promotor

Consensus sequences

Set of most commonly encountered nucleotides at a specific location of RNA or RNA

Y in consensus sequence

Even between pyrimidine (T/C)

R in consensus sequence

Even between purines A/G

N in consensus sequence

Split evenly among all bases

Consensus sequences in bacterial promoters

• -10: 10 bp upstream of start site (closer) TATAAT

• -35: 35 bp upstream of start site (farther) TTGACA

  • Holoenzyme recognizes consensus sequences in bacterial promoters

Third Step of Transcription Initiation

Holoenzyme changes conformation, unwinds the DNA and forms transcription bubble (includes promoter but sigma factor gets pushed away from the promoter and is not included in the transcription bubble)

Fourth Step of Transcription Initiation

Addition of 1st RNA nucleotide, complementary to DNA template

Last Steps of Transcription Initiation

1. RNA nucleotide added to the 3’ end of growing RNA molecule

2. Sigma factor is released as RNA polymerase moves beyond the promoter (sigma factor never cleaved)

Transcription Bubble

DNA strands transiently separated to expose template strand, includes the short DNA sequence base paired w/ 3’ end of RNA

Transcription Pausing

certain DNA sequences or RNA secondary structures can cause RNA polymerase to pause
- incorporation of incorrect nucleotide can also cause RNA polymerase to pause, back up a little, cut off the last few nucleotides from RNA, and then continue transcription

Transcription Termination

2 ways: Rho-dependent (requires Rho protein) and Rho-independent (intrinsic process that does not require additional protein factors)

Rho-dependent Transcription Termination

• RNA sequence contains a Rut Site (Rho UTilization)

• RNA polymerase pauses at a down stream sequence once it senses Rho

• Rho factor moves 5’ to 3’ along RNA

• When Rho catches up w/ RNA and is inside of the transcription bubble, rho uses its helicase activity to separate the RNA-DNA complex and release RNA

Terminator Region in Rho-independent Transcription Termination

Inverted repeats followed by >6 A-Ts → causes intramolecular base pairing when RNA is transcribed

Steps of Rho-independent Transcription Termination

• RNA transcribed from inverted repeats forms hairpin

• Hairpin in RNA causes RNA polymerase to pause

• This destabilization of the DNA-RNA pairing (hairpin is so heavy) → RNA separates from DNA template

RNA polymerases in ALL eukaryotes

RNA polymerase I, II, and III

RNA polymerases ONLY in plants

RNA polymerase IV, V

Basal Transcription Apparatus in Eukaryotes

RNA polymerase + Transcription Factors (TF)

Transcription Factors

proteins that bind DNA sequences and affect levels of transcription

RNA Polymerase II Promoters

Core Promoters - conserved sequences are binding sites for General Transcription Factors (TATA box)

Transcription Initiation in Eukaryotes

Regulatory promoter, core promoter, transcription start site

1. TFIID binds to TATA box in core promoter

2. transcription factors + RNA polymerase II + core promoter (everything binded) - RNA polymerase positioned over core promoter

3. Transcription factors bind to sequences in enhancers

4. DNA loops out, allowing the proteins bound to the enhancer to interact w. the basal transcription apparatus (everything binded) - DNA unwound to form transcription bubble

5. Transcription factors bind to sequences in the regulatory promoter and interact w. the basal transcription apparatus through the mediator - basal TFs stay at promoter & RNA pol II moves downstream

RNA polymerase I

requires termination factor (similar to rho in bacteria)

RNA polymerase III

transcribes terminator sequence (similar to rho-independent mechanism in bacteria)

RNA polymerase II

does not occur at specific sequences, Rat1 exonuclease mechanism

Rat1 Transcription Termination

1. RNA pol II continues mast the sequences that will be in mRNA

2. RNA cut to create a 3’ end in the middle

3. Rat1 is 5’ to 3’, so it binds to 5’ end and goes up to RNA pol II and once it goes up, transcription terminates (rest of RNA before cut degrades)

Polypeptide Chain Connection

N terminus: Free amino group (+ charge), C-terminus: free carboxyl group (C-)

Neurospora (filamentous fungus)

• can grow in rich media (all amino acids supllied)

• or minimal media (no amino acids)

  • synthesize all required amino acids and other essential biomolecules using enzymes

Isolation of auxotrophic mutants

• auxotrophic = unable to produce a particular nutrient

• key to success = main vegetative part of fungus is haploid

  • mutant grows on media w/ arginine

    • mutant must be defective for arginine production

Arginine synthesis

precursor — 1 —> ornithine — 2 —> citrulline — 3 —→ arginine

Block step 1

auxotrophic mutant cannot grow on a media supplemented w/ precursor but can grow on one w/ intermediate orithine, citrulline, or arginine

Nucleotides to Amino Acid

Codon - set of how many every nucleotides

4^# of nucleotides = # of amino acids possible