BIO 221 exam 1

molecular biology

types of weak chemical interactions (weak bonds)

• electrostatic interaction

• h bond

• hydrophobic interactions (hydrophobic groups pushed together)

• van der waals forces

components of an amino acid

• amino group

• carboxyl group

• a-carbon

• side chain

types of amino acids based on R group

• nonpolar

• uncharged polar

• acidic

• basic

disulfide bridge

SH oxidizes

stabilizes 3^o or 4^o structure

only in transmembrane or secreted proteins

uncharged polar amino acids

• side chains include hydroxyl (OH) or amide (CONH2) groups

• hydrophilic

acidic amino acids

• negatively charged

• side chain includes carboxyl group

• hydrophilic

• can form ionic or h bonds

basic amino acids

• positively charged

• side chain includes amino group

• hydrophilic

• can form ionic or h bonds

special amino acids

• glycine

• proline

• cysteine

glycine

• r group is just singular H atom

• lots of conformational freedom (flexible)

proline

• r group bends around to form a ring by covalently bonding to alpha carbon

• rigid. less conformational freedom

• helix breaker

cysteine

• r group can form disulfide bridges (SH)

peptide bond

• covalent linkage between amino acids

• carboxyl to amino

• rigid - partial double bond character

• always written N → C

how do polypeptides adopt diff conformations if peptide bond cant rotate?

two other bonds can rotate:

• phi (ϕ) - between N and alpha C (left)

• psi (ψ) - between alpha C and carboxyl (right)

conformation

change spatial arrangements by rotation

configuration

to change you need to break covalent bonds

protein

polymer(s) of amino acids that is functional

linkers

• transitional regions between 2° structures

• turns and loops

• very flexible

turns vs loops

• turns allow for “reverse” direction (dramatic change in direction) - short

• loop = “alternation” in dimensionality - longer section

hairpin turn (protein)

2 strands connected by a 2 residue turn

α helix

• usually right handed

• stabilized by h bonds

• about 3.6 residues per turn

• r groups pointed out

β pleated sheet

• backbone nearly fully extended - maximize h bonds between strands

• anti parallel or parallel

• side chains point up or down

• curve

coiled coil

supercoiling of α helices around each other (motif)

• nonpolar side chains pointed inward

• amphipathic

• no intercolation

common representations of proteins

backbone, ribbon, cartoon, wire, space-filling

letter code for amino acids in peptide

letter = one letter code of AA

number = position (# from N terminus)

if there is a second letter it shows what the AA was mutated to

motif

recognizable folding pattern of 2 or more 2° structures

about 1000 diff motifs

ex: helix-turn-helix

domain

independently stable region of a protein associated with a particular function. can have multiple motifs or none

modular construction of proteins

idea that new proteins w diff functions can come from diff domain combinations

ligands

molecules that bind to proteins

include small molecules, other proteins, other macromolecules

allosteric regulation

ligand binds, changing the conformation of the protein, affecting its function

• changes affinity for second ligand

• regulate protein activity

• reversible binding (noncovalent)

• usually small molecule

positive and negative regulation

covalent modification

• post translational

• modification of AAs to change properties of protein

  • phosphorylation

  • acetylation

  • methylation

  • glycosylation

  • lipidation

  • addition of other proteins

disordered regions of proteins

• unstructured

• some are flexible hinges between domains

• some allow promiscuous interaction

IDPs (intrinsically disordered proteins)

• completely structurally disordered

• functional promiscuity

• scavengers: serve as reservoirs for ions or small molecules in solution

• scaffolds that allow multiple other proteins to bind

number of bp in one turn of a dna double helix

10.5 bp

purines

guanine and adenine

pyrimidines

cytosine and thymine

uracil structure

pyrimidine

thymine without the methyl

two c=o

cytosine

pyrimidine

one C=O and one amine

thymine

pyrimidine

two c=o and one methyl

adenine

purine

one amine

guanine

purine

one amine and one c=o

composition of a nucleotide

sugar backbone (pentose)

phosphate

nitrogenous base

base flipping

• with ATP, bases can flip out of the helix

• necessary during repair and recombination

DNA transactions examples (protein-dna)

• DNA replication

• transcription of DNA

• DNA packing by histones

• DNA repair

code that proteins read in DNA double helix

A = H bond acceptors

D = H bond donors

H = nonpolar H (van der waals)

M = methyl groups (hydrophobic forces)

major and minor groove

major groove contains more information than the minor groove (can recognize A:T vs T:A)

minor groove not as accessible

B DNA

• most common

• right handed

• high humidity

A DNA

• low humidity or nonaqueous solutions

• more compact

• right handed

• thought to help protect against damage

Z DNA

• left handed

• zigzag - bases arent straight

• thought to be involved in gene regulation

• formed to provide relief from supercoiling (torsional strain) while transcription occurs

• formation can also signal DNA damage or instability

DNA double helix is never perfect

propeller twist - base pairs not in same plane

precise rotation per bp is not consistent

what denatures DNA?

heat - thermal energy disrupts h bonds

alkaline solution - OH destabilizes h bonds

formamide and urea - same

renaturation and reannealing

DNA from same source

DNA hybridization

annealing of ssDNA from two different sources

could be DNA and RNA

don’t need 100% complementarity to hybridize

hyperchromicity

increase in light absorbance (260nm) when DNA goes from ds to ss

the curve is steep at the melting point (Tm)

what influences Tm?

G:C content (more G:C = higher Tm because they are 3 h bonds)

higher ionic strength = higher Tm

alternation

when DNA is linear and then circular or the other way. ex: lambda phage is a dna virus of e coli that has linear dna that circularizes once it is injected into the bacterial cell

negative supercoil

• underwound (fewer bases per turn)

• constrained dna is usually in this form

• easy to separate strands - good for replication and transcription

• nucleosomes package dna to introduce negative supercoils

positive supercoils

• less common than negative

• overwound (more bases per turn)

• resists unwinding of the helix - may be an adaptation to high heat

topoisomerase i

• nicks DNA: only cuts one strand

• adds and subtracts twists one at a time

• then ligates ofc

topoisomerase ii

• cuts both strands of DNA

• requires ATP to rotate the DNA

• adds or subtracts two twists at a time

• then ligates

protein family

proteins with similar primary and tertiary structure and function

protein superfamily

two or more families with a little primary similarity but use same motifs and often have similar functions

DNA topoisomers

same chemical formula, different conformation because the strands are wrapped differently

can be separated by gel electrophoresis

EcoRI effect on supercoiled plasmid DNA

linearizes the plasmid DNA

DNAse I effect on supercoiled plasmid DNA

relaxes all supercoils but doesn’t religate (quick)

Topoisomerase effect on supercoiled plasmid DNA

relaxes supercoils and religates, but works slowly so it depends on the treatment time how much of the plasmid gets relaxed

RNA structure/properties

1. usually single stranded

2. Uracil not thymine

3. ribose not deoxyribose in the sugar-phosphate backbone

4. can naturally form complex structures by folding

stability of RNA

less stable than DNA because:

• autocatalytic degradation (thru hydrolysis of backbone)

• cytosine can undergo spontaneous deamination to become uracil (and its difficult for the cell to know that has happened)

• uracil can mispair

• uracil is more sensitive to UV than thymine

RNA function: genetic intermediate

• mRNA

• between gene and protein

RNA function: structural role

• rRNA

• structural component of ribosome (also enzymatic)

RNA function: adaptor role

• tRNA

• adaptor between codons and amino acids

RNA function: regulatory role

• miRNA

• gene regulation (where and when a gene product will be made)

RNA function: enzymatic role

• ribozyme

• usually involved in cleavage of other nucleotides

• catalyzes addition of AAs in ribosome

RNA secondary structures

• hairpin (5-10 NT loop at end)

• bulge (one side)

• internal loop (basically double bulge)

• stem loop (>10 NT end loop)

• junction (helices diverging from a single point)

• pseudoknots (pairing between bases that are not contiguous - stabilizing)

• tetraloop (base stacking - stabilizing)

benefits of non-Watson-Crick pairing in RNA

• more self complementarity

• stabilization

• adds complexity to the structure

examples of ribozymes

• RNA splicing - removing introns from certain mRNAs requires ribozymes

• RNAse P - first ribozyme discovered. processes large tRNA precursor into tRNA

• most ribozymes cleave other RNAs

• some cleave themselves (hammerhead ribozyme)

evidence for RNA world hypothesis

RNA can serve as both a repository of information and as a catalyst

RNA recognition motif (RRM)

• 4 stranded antiparallel beta sheet with 2 alpha helices

• many RNA binding proteins have this motif

• surface of the sheet mediates the interaction

  • specifically 3 conserved residues

chromatin

the material that collectively composes the chromosome. compacts, protects, organizes DNA

histones

proteins that are major players in packaging DNA

• small

• positively charged

• similar across eukaryotes

• core and linker histones

nonhistone proteins

not as abundant as histones

regulate DNA packing, transcription, repair, replication, and recombination

nucleosomes

nucleosome core particle = NPC/core

8 histones in core and 1 linker histone

about 147 bp wrapped around core, 20+ in between cores

DNA wrapped around core in a left handed way - stabilize negative supercoils

histone names

H2A, H2B, H3, H4 = core

H1 = linker

structure of core histone

3 alpha helices separated by loops and an N-terminal tail that sticks out of the nucleosome

importance of N-terminal tails of histones

• stabilize wrapping of DNA around nucleosome

• highly regulated - can undergo methylation, phosphorylation, and acetylation that alters chromatin accessibility and recruits modifiers

structural basis for binding and bending DNA

• 14 contact sites b/w nucleosome and dna on the minor groove

• 142 h bonds b/w them

  • mostly to backbone, some to bases

  • h bonding facilitates bending because of the charge stabilization

heterochromatin

• dense

• limited expression

• dark stain

• associated with specific regions on chromosome (centromere and telomere)

eucromatin

• open

• potentially active

• poor stain

• less organized

• higher expression

linker histone (H1)

binds to linker DNA and middle of core DNA

further tightens association- more compact

turns 10nm fibers into 30 nm

solenoid/superhelix model

• structure of 30 nm fiber

• flat surfaces of histones are adjacent

• linker DNA buried

• hole in the middle

zigzag model

• structure of 30nm fiber

• linker dna passes thru central axis (no hole)

• exit and entry points more accessible

looped domains

• further compaction of chromatin

• 30 nm fiber → 700 nm fiber

• large loops and coils and folds

• the width of a whole chromosome is 1400 nm

regulation of chromatin structure (chromatin remodelling)

• overall to either condense or open up genomic dna to control gene expression

• tails of histones get modified by enzymes

acetylation enzymes (histone modification)

• histone acetyltransferase (HAT) +

• histone deacetylase (HDAC) -

phosphorylation enzymes (histone modification)

• histone kinase +

• histone phosphatase -

methylation enzymes (histone modification)

• histone methyltransferase +

• histone demethylase -

takeaways from histone tail modifications

• type of mod and position are important

• acetylation = transcriptional activation

• methylation = transcriptional silencing

what proteins recognize the histone tail modifications?

• nucleosome-modifying enzymes (complexes)

• nucleosome-remodeling complex

nucleosome modifying enzymes

• recognize modifications and can add modifications (any)

• some modifications can change chromatin structure, others recruit other proteins to do so

nucleosome remodeling complex

• facilitate nucleosome movement (using ATP)

• mediate the following:

  • sliding - movement along dna

  • transfer or ejection

  • dimer exchange - histone dimers are replaced with variants (numerous variants exist, ex: CENP-A)

CENP-A

replaces H3 in nucleosome of centromere with one with an extended tail that can attach to kinetochore proteins