Cell and Molec Exam 1

What are micropipettors?

a “sophisticated eyedropper,” transfers microliters of fluid

What micropipettor should be used for a given volume?

p10 → 1-10microliters

p20 → 2-20microliters

p200 → 20-200microliters

p1000 → 200-1000microliters

What are plasmids?

plasmids are small, circular, double-stranded DNA molecules that are distinct from a cell’s chromosomal DNA

naturally exist in bacterial cells

How are plasmids isolated in the lab?

cells are separated out/lysed

then centrifuged, plasmid is suspended in a supernatant

you mix the supernatant with binding buffer to increase salt concentration and improve binding

then the column captures the plasmid

you use a wash to “clean” the plasmid

then you use elution buffer which releases the bound plasmid from the column

and then it’s measured by the nanodrop

How can the concentration of DNA be measured experimentally?

by using a nanodrop at 260nm

What do the different wavelengths on the Nanodrop measure?

260nm → quantifies the nucleic acid

280nm → measures for protein contamination/protein quantification

230nm → measures the background absorbance for organic and salt contamination

What are the components of a plasmid? How do the different components of a plasmid contribute to function?

components of plasmids:

an ori (origin of replication) → where DNA replication initiates

antibiotic resistance gene → a way to select for bacteria with plasmid

multiple cloning sites → a small region with many restriction enzyme sites

an insert → the DNA you are recombining into the plasmid

the primer binding sites → a DNA sequence that is helpful in sequencing the insert

reporter gene - usually an enzyme, often provides a way to visually screen if the experiment is successful

How are plasmid maps constructed?

plasmid maps are built by measuring DNA fragments after cutting the plasmid with restriction enzymes

What are different ways and approaches to study cells?

microscopy, cell culture, biochemical analysis

What are a few advantages of including non-covalent bonds in biological macromolecules?

noncovalent bonds give macromolecules shape and allow them to bind with other selected molecules. the ribosome is a good example of this

these bonds account for a good chunk of the dinstinctive chemistry responsible for making life possible

What are the major molecular components of cells?

macromolecules such as lipids, proteins, DNA/RNA, and polysaccharides

How are macromolecules assembled?

macromolecules are assembled from subunits, monomers, which bind to each other chemically

What types of bonds hold together carbohydrates? Proteins? Nucleic acids?

polysaccharides: glycosidic bonds between sugars

proteins: peptide bonds between amino acids

nucleic acids: phosphodiester bonds between nucleotides

How are phospholipid subunits held together? How do unsaturated bonds in phospholipid fatty acids change the structure of the fatty acid?

a glycerol backbone attached to two fatty acid tails via ester bonds and a phosphate group via a phosphoester bond

an syn unsaturated bond is a C=C, and that creates a kink in the tail due to the hybridization of the carbons changing to sp²

How might changes in monomer composition influence macromolecule structure?

the specific properties, sequence, and bonding arrangements of monomers/subunits influence how a macromolecule folds, branches, and interacts with its environment

How do macromolecules assemble to make larger cellular components such as organelles?

via noncovalent bonds/interactions

What type of bonds hold together the bases in each strand of DNA?

phosphodiester bonds: strong covalent bonds link the 5' carbon of one sugar molecule to the 3' carbon of the next sugar molecule via a phosphate group

What type of bonds hold together the complementary base pairs in the double helix?

Weak hydrogen bonds

A-T: 2 bonds

G-C: 3 bonds

What is the difference between heterochromatin and euchromatin?

euchromatin (The "Active" DNA): has a loosely packed, open structure. 60% of typical interphase chromosome. cell's machinery can easily access the DNA, the genes here are actively being read and used (transcribed) to make proteins.

heterochromatin (The "Silent" DNA): has a densely packed, closed structure. 40% of typical interphase chromosome. cell's machinery physically cannot get in to read the genes. these genes are turned off/silenced

What are chromosomes?

a thread-like structure made of DNA and proteins packed tightly inside the nucleus of living cells

humans have 46 chromosomes, 23 pairs in each cell

What are the components of a chromosome?

chromosomes are composed of:

DNA

Histones

Centromeres

Telomeres

How is DNA compacted to fit within the nucleus? How do histones help organize the DNA and prevent tangling?

DNA wraps almost 2x around a core of eight histone proteins to form a nucleosome. nucleosomes create a chromatin fiber that reduces the length of the DNA significantly.

a linker histone (H1) binds to the stretches of DNA between nucleosomes, pulling them together into a denser, stacked structure

the fiber folds into large loops attached to non-histone protein scaffolds

during cell division, these loops condense further into tight, manageable chromosomes.

What are the roles of cohesins and condensins? How are these proteins similar? How are they different?

Both are SMC ring complexes (SMC proteins that use ATP hydrolysis energy to motor along DNA) that use looping and tethering to organize chromosomes

Cohesins (TETHERS): organizes the structure of interphase chromosomes, performs inter-molecular interactions by connecting separate sister chromatids.

Condensins (LOOPS): performs intra-molecular interactions, looping and compacting a single DNA molecule back onto itself. replaces most of the cohesins as the cells prep to divide (metaphase), forms loops of their own to wind the chromatin even tighter.

How are genes expressed into functional RNAs and proteins?

via transcription and translation

How is RNA structurally similar to DNA?

both are nucleic acid polymers built up of repeating of nucleotides that have a phosphate backbone design and nitrogenous bases

What are important differences between the structures of RNA and DNA?

RNA is single-stranded and contains uracil instead of thymine

What factors determine the structure of an RNA molecule?

singe-stranded and has nucleotide sequences

→ complementary basepairs on the same RNA molecule can interact with each other and fold the molecule into a different shape, and unconventional basepair interactions can fold it even further

What different types of RNA are produced by the cell?

there is mRNA, rRNA, siRNA, miRNA, tRNA, lncRNA

What is a promoter? Why is it essential for RNA transcription?

a promoter is a gene region on the RNA strand that acts as a signal for the RNA polymerase to attach and begin transcribing.

without a promoter, transcription cannot begin.

What factors are needed for transcription initiation in eukaryotes?

transcription initiation in eukaryotes requires three different types of RNA polymerase which transcribe different kinds of RNA

it also needs: consensus sequence, a set of general transcription factors, a collection of activator proteins, and elongation factors which bind to the promoter and position the RNA polymerase

How are eukaryotic and prokaryotic transcription initiation similar and different?

similar: both need a consensus sequence and RNA polymerase to bind to a specific DNA promoter region and start making RNA

eukaryote only: have three RNA polymerases, have one main promoter (TATA), use general transcription factors and accessory proteins and elongation factors

prokaryote only: have only 1 RNA polymerase, have 2 main promoters (-10 box, -35 box), use sigma factor

Why is it essential for cells to regulate transcription levels?

because they need it to control which proteins are made, conserve energy, and respond to changes in their environment

What are important features of regulatory DNA sequences?

repressor, activators, and promoters

DNA binding proteins which can bind to the strand and impact specific segments of DNA.

control when, where, and how much genes are expressed.

What is an operator?

an operator is a short DNA sequence that is recognized by the transcription regulator, and is bound to when gene doesn’t need to be expressed

How do activators and repressors work to control gene expression?

activators: turn genes on/up, CAP protein binds to binding site, activates transcription

repressors: turn genes off/down, repressor binds to operator, inhibits transcription

How is the activity of transcription factors changed by small molecules such as inducers and corepressors?

small molecules change the activity of transcription factors by acting as allosteric effectors

when an inducer or corepressor binds to a transcription factor (TF), it induces a conformational change that fundamentally alters the protein's ability to interact with DNA or recruit other regulatory cofactors

How are eukaryotic RNAs processed? Where do these processes occur?

eukaryotic mRNAs are processed inside of the nucleus.

large molecules called spliceosomes cut out the noncoding sequences (introns) and then send the mature mRNA out of nucleus into cytoplasm to be translated

What are introns? What are exons?

introns are “nonsense” gene sequences that don’t code for anything

exons are gene sequences that code for proteins

What factors contribute to mRNA splicing? Be able to explain what would happen if one of these factors was missing or mutated

the 5’ GU and the 3’ AG which direct the spliceosome’s splicing

if missing it could mess up expression and result in lack of a protein essential for function

How does splicing contribute to phenotypic diversity in organisms? Be able to explain why splice variants produce different versions of translated proteins.

enables a single gene to encode multiple distinct mRNA transcripts and protein variants

if something splices differently then have a different codon, and that leads to different proteins

Why is processing necessary prior to translation?

to stabilize the messenger RNA molecule, remove non-coding sequences, and ensure the cell produces accurate, functional proteins

What is a riboswitch?

a regulatory segment of a messenger RNA (mRNA) molecule that binds to a small target molecule to directly control its own gene expression. They have an aptamer domain and expression platform

What are the similarities and differences between miRNAs and siRNAs? Be able to make a prediction about whether a small RNA is acting as an miRNA or an siRNA based on data.

miRNAs - microRNA, matches multiple different mRNA transcripts with only partial sequence pairing, endogenous (produced by cell)

siRNAs - small interfering RNAs, have exact complementary pairing to target sequence, exogenous (like from outside sources like viruses)

both are short non-coding RNA that serve as gene expression regulators, silencing expression

How do small RNAs provide protection against viral infections? For example, be able to describe the roles of RNA in CRISPR in bacteria.

act as precise molecular guides that direct protein machinery to find and destroy the genetic material of invading viruses

siRNA direct indentification and destruction of viral genomes. they provide a record of past infections for bacteria by taking fragments of each inside and storing it of the bacteria genome called the CRISPR locus, which help protect them from future infections

What are roles of lncRNAs in the cell?

long noncoding RNAs can fold into specific three-dimensional structures through complementary base paring that can serve as scaffolds which promote the interaction of proteins that function together in a complex

How did scientists crack the genetic codes (translation)?

Nirenberg and Matthaei

through an experiment by which they broke open Ecoli cells to use their cytoplasm and ribosomes for protein synthesis without living cells and added synthetic mRNA made entirely of uracil, which produced a protein made entirely of a single amino acid which proved that UUU codes for phenylaline

What is wobble? How does alternative base pairing contribute to wobble? Be able to explain why wobble has an effect on the impact of mutations.

wobble refers to the flexible base-pairing rules between the third nucleotide of a mRNA codon and the first nucleotide of a tRNA anticodon

single tRNA molecule can recognize and bind to more than one specific codon, explaining why cells do not need a unique tRNA for every single one of the 64 amino acid-coding codons

What are the three types of RNAs essential for translation?

tRNA, rRNA, and mRNA

How are amino acids loaded onto the correct/corresponding tRNA?

Adaptor molecules bind to a codon with one part and to an amino acid with the other

What are the roles of rRNAs in translation?

The mRNA is read 3 bases at a time, and the tRNA is fixed with the corresponding protein

Why is the ribosome considered a ribozyme? What components of the ribosome catalyze the peptide bond?

because they double as a organelle and a catalyst making amino acid bonds. is a complex of rRNA and ribosomal proteins

ribosomal proteins help fold and stabilize the RNA core while permitting change of rRNA conformation that are necessary for RNA to catalyze efficient peptide bonds

basically, ribosomes have catalytic core that makes peptide bonds of amino acids

How is translation initiated? How does the ribosome assemble.

translation is initiated by the translation initiation factors (t.i.f) and the special initiator tRNA (which always carries methionine)

the ribosome starts with a small ribosomal subunit, which t.i.f’s and special tRNA bind to, and then they attach to mRNA, and large ribosomal subunit binds on top after degradation of t.i.f

What are the steps of translation that are repeated during synthesis of a protein?

the elongation stage is repeated

codon recognition by tRNA, matches anticodon → peptide bond links new amino acid to the growing polypeptide chain → ribosome moves forward by one codon on the mRNA → repeat

What is a stop codon? How does it signal for the end of a polypeptide?

the stop codon are UAG, UAA, or UGA

release factors bind to the A site of a ribosome and signal it to break off the polypeptide chain and dissociate once it reaches the UAG

How are prokaryotic and eukaryotic ribosomes similar? How are they different? Why is this important for the development of antibiotics?

Prokaryotic and eukaryotic ribosomes both consist of subunits, a large and small. But the eukaryotic ribosome is 80S (60S and 40S) and the prokaryotic ribosome is 70S (50S and 30S)

What does a chromosome need to replicate?

to replicate, a chromosome needs:

an ori → the site where replication begins, a specialized nucelotide sequence

telomeres → DNA sequence that marks the ends of each chromosome, serve as protective cap to keep the tips from being recognized as broken DNA in need of repair

centromere → another special DNA sequence that allows duplicated chromosomes to be separated during the M phase

what does a chromosome need to divide?

needs centrosomes and kinetochores

kinetochores:

What is a consensus sequence?

a common nucleotide sequence or amino acid sequence found in highly conserved regions of DNA or RNA or proteins.

consensus sequence is simply the "most common version" of a specific stretch of DNA, RNA, or protein. If your DNA varies too much from the consensus, the cell might not recognize the signal, and things can go wrong.

What makes up the spliceosome?

the spliceosome is made up of snRPs, small nucleic ribosomal proteins