Cell Bio Exam 4

Ch. 19

Ribosomes

Carry out protein synthesis

Consists of large and small subunit (dissociable, contian variable amount of protein and rRNA)

In cytoplasm of prokaryotes and eukaryotes; in the mitochondria and chloropplasts as well

tRNA

align amino acids in the correct order along the mRNA template

Aminoacyl-tRNA synthetase

attach amino acids to their appropriate tRNA molecule

mRNA

messenger RNAs - encode the amino acid sequence information

Contain coding region and 5’ + 3’ UTR

Protein factors

facilitate several steps in the translation process

A (aminoacyl) Site

binds each newly arriving tRNA with attached amino acid

P (peptidyl) Site

where tRNA carrying the growing polypeptide chain resides

E (exit) Site

from which tRNAs leave the ribosome after they have discharged the amino acid

mRNA binding site

binds a specific nucleotide sequence near the 5’ end of mRNA, placing the mRNA in proper position for translation of its codonCod

Coding region (mRNA)

provide the information for the synthesis of the correct order of amino acids

Start Codon

typically AUG

Stop Codon

UAG, UAA, UGA

Eukaryotic mRNAs

contain a 5’ cap and 3’ poly A tail, which are part of the 5’ UTR and 3’ UTR respectively

Prokaryotic mRNAs

contain a ribosome binding site (Shine-Dalgarno Sequence)

Genetic Code

set of rules by which information encoded in genetic material (DNA or RNA sequences) is translated into proteins (amino acid sequences) by living cells

Triplet code

a code in which three base pairs in double-stranded DNA are required to specify each amino acid in a polypeptide

Degenerate Code

a give amino acid can be specified by more than one codon

Nonoverlapping code

each nucleotide is a part of one, and only one, triplet

tRNA attachment to amino acid

two kinds of specificity:

1. Each tRNA binds one specific amino acid

2. Each recognizes on eor more mRNA codons that are specific to an amino acid

-Linked to their corresponding amino acid by an ester bond that joins the amino acid to the 3’ OH group fo the adenine nucleotide located at the 3’ end of ALL tRNA molecules

-Aminoacyl-tRNA synthetase: enzyme responsible for catalyzing formation of enzyme bond between correct amino acid and tRNA (covalent linkage, 20 aminoacyl-tRNA synthetase for 20 amino acids)

Translation Initiation

componets of translational apparatus come together with an mRNA, and a tRNA carrying the first amino acid binds to the start codon

Translation Elongation

Amino acids are brought to the mRNA by tRNAs and are added, one by one, to a growing polypeptide chain (in the N-terminus to C-terminus direction)

1. Aminoacyl tRNA binding

2. peptide bond formation (catalyzed by peptidyl transferase)

3. Translocation (A site to P site to E site)

   1. Exception: Initiator tRNA binds to P site directly

Termination

a stop codon (UAG, UAA, UGA) in mRNA recognized by protein release factor at the A site, and hydrolysis of GTP accompanies release of completed polypeptide; tRNA, mRNA, ribosomal subunits, and release factors then dissociateE

Euakaryotic Translation Features

differences mostly confined to initation stage:

1. AUG start codon in eukaryotes (and archaea) specifies the amino acid methionine rather than N-formylmethionine in bacteria

2. Eukaryotes use different set of initiation factors known as eIFs (about a dozen proteins)

3. A common start sequence in eukaryotes is ACCAUGG (kozak sequence), where underlined triplet is actual start codon

Molecular Chaperones

Facilitate polypeptide folding; several are often required and they act in sequence

Bind polypeptide chains during the early stages of folding

Mutations and Translation

Inerstion, Deletion, Duplication, Inversion, Translocation (reciprocal)

Posttranslational Processing

Eukaryotes: methionine at the N-terminus is removed

Whole blocks of amino acids can be removed from the polypeptide (e.g. insulin)

Chemical modification of amino acids - methylation, phosphorylation, acetylation, glycosylatoin - common

Protein Targeting and Sorting

mechanism by which a cell transports proteins to the appropriate positions in the cell or outside of it

eukaryotic compartments:

1. Endomembrane system: ER, Golgi Apparatus, lysosomes, secretory vesicles, nuclear envelope, plasma membrane

2. Cytosol

3. Mitochondria, Chloroplasts, peroxisomes, interior of nucleus

Polypeptide Synthesis

Begins in cytosol, but takes on of two alternative routes when polypeptide is about 30 amino acids long

Cotranslational Import

for polypeptides destined for endomembrane system, or for export from the cell - transferred across/into the ER membrane as they are made

posttranslational import

for polypeptides that either remain in cytosol or that are imported into nucleus, miochondria, chloroplast, or peroxisomes (after synthesis in cytosol)

Proteins are synthesized on free ribosomes, released into cytosol, and each protein carries a targeting signal that directs it to correct organelle

Posttranslatoinal import of polypeptides into the Mitochondrion

Transit sequence

Membrane receptor:

-TOM —Translocase of the Outer Mitochondrial membrane

-TIM — Translocase of the Inner Mitochondrial membrane

Chaperones: keep the polypeptide partially unfolded after synthesis, drive the translocation itself, help the polypeptide fold into its final confromation

Cotranslational Import Mechanism - Soluble Protiens

SRP - Signal recognition particle

Polypeptides destined for endomembrane system contain an ER signal sequence; enter the ER as being synthesized

1. SRP binds to ER signal sequence and blocks translation

2. SRP binds to SRP receptor; ribosome docks on membrane

3. GTP binds to SRP and SRP receptor, pore opened and polypeptide is inserted

4. GTP is hydrolyzed and SRP is released

5. Signal sequence is cleaved by signal peptidase as polypeptide elongates and translocates into ER lumen

6. completed polypeptide is released into ER lumen, ribosome is released, and translocon pore closes

Cotranslational Insertion Mechanism - Membrane proteins

membrane protines destined for endomembrane system contain a stop-transfer sequence or internal star-transfer sequence in addition to an ER signal sequence

Polypeptide is inserted inthe ER membrane as it is being synthesized

Ch. 20

Constitutive Genes

those genes that are expressed all the time

Regulated Genes

those genes whose expression is regulated so that the amount of the final product - protein or RNA - is carefully tuned to the cell’s need for that product (most genes)

Galactoside permease

responsible for transporting lactose into cell

B-galactosidase

catalyzes the breakdown of lactose

synthesis of B-galactosidase turns on in presence of lactose and turns off in absence of lactose

Substrate induction

Enzyme synthesis is induced by its substrate

Enzymes regulated this way = inducible enzymes

Characteristic of most catabolic pathways in bacteria

Operon

group of genes with related function that are clustered together with DNA sequences that allow the genes to be turned on and off simultaneously; common in prokaryotes but not in eukaryotes

polycistronic mRNAs

encode more than one polypeptide

Lactose Catabolism

involves two types of genes

1. genes coding for enzymes involved in lactose uptake and metabolism: lacZ, lacY and lacA

2. Regulatory gene whose product controls the activity of the first set of genes: lacI

lacZ

beta-galactosidase

lacY

galactoside permease

lacA

trans-acetylase

lacI

encodes the repressor protein - turns off expression of downstream genes

End product repression

enzyme synthesis is repressed by its end product

characteristic of most biosynthetic pathways inbacteria (trp Operon)

regulation of Tryptophan Operon

Tryptophan present - activates repressor -repressor binds to operator - operon repressed

Tryptophan absent - repressor remains inactive - repressor does not bind to operator - operon active

Genomic Control

regulatory change in the structural organization of the genome: gene amplification, gene deletion, DNA rearrangements, chromatin decondensation (unfolding), Histone modificaiton: methylation and acetylation

Transcriptional Control

interaction of general transcription factors with the core promoter often initiates transcription at low rate

DNA control sequences:

1. Proximal control elements - upstream of core promoter, but w/in 100-200 bp of it

2. Enhancers and silencers - upstream or downstream of genes, often far away from promoter

Regulatory Transcription Factors:

1. Activators - bind to enhancers and activate transcription

   1. Repressors - bind to silencers and reduce transcription

Core promoter

TATA box and Inr - where general transcription factors and RNA polymerase assemble for the initiation of transcription

Proximal Control Elements

within about 100 nucleotides upstream from core promoter lie several proximal control elements - stimulate transcription of the gene by interacting with regulatory transcription factors

Enhancers and Silencers

can function at same level at distances up to several hundred kb away

repressors can compete with activators for binding to enhancers, or they can bind to activators to imped their activity

Enhancers can be upstream, downstream, and distance not important

RNA processing

alternative splicing - exons can be either excluded or included from a pre-mRNA resulting in multiple mRNA isoforms

Translational Control

translation rates can be controlled by translational repressors - proteins that selectively control translation of a particular mRNA by binding to 5’ UTR or 3’ UTR

(allows cells to respond to changes in environment faster than in transcriptional control)

IRE: iron response element

1. Low Iron conc - IRE binding protein binds to IRE - translation of ferritin mRNA inhibited

2. High Iron conc - IRE biding protein cant bind to IRE - translation of ferritin mRNA proceeds

Post translational control

Ubiquitin-dependent protein degradation - Ubiquitin attached to lysine in targeted proteins through sequention actoin of:

1. A ubiquitin-activating enzyme (E1)

2. A ubiquitin conjugating enzyme (E2)

3. a ubiquitin ligase (E3)

   1. Proteasome then degrades the targeted protein into short peptides

Ch. 21

Restriction Enzymes

proteins isolated from bacteria that cut foreign DNA molecules at specific internal sites - restriction sites - 4-6 nucleotides long

Blunt ends

fragments generated from both DNA strands being cut in same location

Sticky ends

fragments generated from DNA strands being cut in a staggered fashion

Gel electrophoresis

allows DNA to be seperated by size

negative charge of DNA moves toward anode

Smaller fragments move rapidly - travel further

Polymerase Chain Reaction (PCR)

uses specific primers and DNA polymerase (Taq) to exponentially amplify specific target DNA fragments (template)

Amplifies DNA fragments through repeated cycles of heating and cooling (thermal cycling)

Steps of a PCR Cycle

1. Denaturation - heat to seperate strands

2. Annealing - cool, allow primer to attach

   1. Extension - Taq adds bases to seperated strands

RT-PCR

complementary DNA (cDNA) can be made from mRNA using reverse transcriptase

In RT-PCR, double-stranded cDNA can serve as template, to be amplified using primers - Can be used to determine whether an mRNA is present in a sample, or quantifying the expression level o foriginal starting mRNA

Southern Blotting

method for detection of a specific DNA sequence from complex DNA samples

Uses a DNA probe - a single stranded DNA that can identify a desired DNA sequence by base pairing with it

Involves seperation of DNA fragments by electrophoresis, transfer of seperated DNA fragments from gel to a filter memrane, and detection of specific DNAs by probe hybridization

Northern Blotting

similar to southern blotting, but RNA is analyzed

electrophoresis seperates RNA samples, RNA transfered to blotting membrane, RNA detected by a hybridization probe

Widely used to determine gene expression

Western Blotting (Immunoblotting)

uses antibodies to detect and quantify a specific protein from complex protein samples

electrophoresis seperates proteins, proteins transfered to a membrane, incubated with an antibody, protein detected by chemiluminescence

DNA sequencing

Determine the linear order of bases in DNA

Sanger Method

DNA sequencing using Dideoxy Chain Termination

Uses dideoxynucleotides to interfere w/ normal enzymatic synthesis of DNA

ssDNA used as template for new comp. DNA

Sequencing by synthesis, high accuracy, long read lengths, small amount of data generated

Automated Sequencing Method

Second generation - sequencing by synthesis, short reads, high accuracy

Third Generatoin - single molecule templates, low accuracy (though improved), long read lengths

Genome Wide Association Studies

investigate multigenic diseases

genomes of thousands of individuals sequenced

Genetic variants in those w/ disease compared to those w/o disease

Associatoin between genetic variants and disease risk requires intensive statistical analysis

Genetic engineering

field opened up by rapid advances in ability to manipulate genes

Requires engineering organisms themselves

Transgenic Organism

Organism that contains a foreign piece of DNA so that it can be passed to subsequent generations

Created by transgenesis

Transcriptional Reporters

used to understand how gene expression is regulated

The coding region of a gene is replaceed with the coding region of a reporter gene

Reporter gene encodes harmless protein but that can be easily seen - allows dissection of the regulatory componets of the gene

Genome Editing

technologies to alter the genomcs of cells and organisms

Specific sequences are used to target complexes to certain genomic sites where genomic DNA is directly altered

Double-stranded breaks induced and repaired

Results in rekmoval of DNA or replacement of normal DNA w/ another sequence

If in germ line - can be transmitted to new generations

CRISPR-Cas9

powerful method in genome editing

Clustered Regularly Interspersed Short Palindromic Repeats

causes break and repairs to create mutation

Ch. 24

Eukaryotic Cell Cycle

M phase involves two overlapping events: nucleus divides first and cytoplasm second

Nuclear Division - Mitosis

Cytoplasm Division - Cytokinesis

Interphase

G1 - cells grow and prepare for DNA replication

S - DNA replication

G2 - cell grows and prepares for mitosis, chromosomes condense into compact, folded structures toward end of G2P

Prophase

when individual chromosomes become visible as discrete objects in the light microscope

Prophase Chromosome: composed of tow chromatids tightly attached to each other

Animal Cells - nucleoli dispere, Plant cells - can remain visible

Two Centrosomes with centriole pairs - function as Microtubule organizing centers - being to migrate away from each other

As centrosomes moe - act as nucleation sites for microtubules destined to form mitotic spindle

Dense starburts of MTs called an aster forms near each centrosome

Prometaphase

marked by fragmentation of the membranes of the nuclear envelope

Centrosomes complete movement to opposite sides of nucleus - spindle MTs contact the condensed chromsomes

MTs attach to chromosomes in the centromere region, a constricted area where the two members of each chromatid pair are held together

Kinetochore - structure that attaches the paired chromatids to spindle MTs

Metaphase

fully condensed chromosomes all align at the metaphase plate

Sister chromatids of each chromosome are being actively tugged toward opposite poles; the forces acting on them are equal in magnitude and opposite in direction

Anaphase

shortest phase of mitosis

Two sister chromatids of each chromosome abruptly seperate and move toward opposite poles

1. Anaphase A - chromosomes are pulled toward spindle pores as kinetochore MTs get shorter

2. Anaphase B - spindle poles themselves move away from each other as polar MTs lengthen

A and B can take place at same time, or B may follow A

Telophase

daughter chromosomes arrive at the poles of the spindle

Chromosomes uncoil into interphase chromatin

Nucleoli reappear, nuclear envelopes reform

Cytokinesis takes place

G1-S Transition (restriction point)

cells that pass through are committed to S phase; those that do not will enter into G0 and reside there until a signal allows them to reenter G1G

G2-M Transition

committment ismade to enter mitosis; some cells can be arrested in G2 indefinitely, and those cells enter a state analogous to G0

Metaphase-Anaphase Transition

commitment i smade to move the two sets of chromosomes into the new cells; before cells can being anaphase, all the chromosomes must be properly attached to the spindle