Test 2 for Cell and Molec

nucleotide subunits within a strand

nucleotide subunits within a strand

held together by phosphodiester bonds that link the 5ʹ end of one sugar with the 3ʹ end of the next

DNA double helix

• held together by hydrogen-bonding between the bases on different strands

  • making all the bases face inwards and the sugar-phosphate backbones face outside

  • the strands run anti-parallel to one another

gene expression:

the process by which the nucleotide sequence of a gene is transcribed into the nucleotide sequence of an RNA molecules and then translated into the amino acid sequence of a protein

“junk DNA”:

large excess of interspersed DNA that doesn’t have a set use

telomeres

mark the ends of each chromosome

contain repeated nucleotide sequences that are required for the ends of the chromosomes to be fully replicated

also serve as a protective cap that keeps the chromosome tips from being mistaken by the cell as broken DNA

centromere

• specialized DNA sequences that allows duplicated chromosomes to be separated during M phase

  • DNA coils up and become more compact and condense to form mitotic chromosomes

  • once condensed the centromere allows the mitotic spindle to attach to each duplicated chromosome in a way that directs one copy of each chromosome to be segregated to each of the two daughter cells

histones

present in enormous quantities, and their total mass is equal to that of the DNA itself

nonhistone chromosomal proteins:

present in large quantities, include hundreds of different chromatin-associated proteins

chromatin:

complex of both classes of proteins with nuclear DNA that compacts DNA into a denser structure

nucleosome

nucleosome: convert DNA molecules in an interphase nucleus into a chromatin fiber that is 1/3 the length of the initial DNA

histone octamer:

an individual nucleosome core particle that consists of a complex of eight histone proteins

ways to adjust rapidly to the local structure of their chromatin

• using a set of ATP-dependent chromatin-remodeling complexes

  • protein machines use the energy of ATP hydrolysis to change the position of DNA wrapped around nucleosome

  • by interacting with the histone octamer and the DNA wrapped around the octamer, the chromatin complexes can locally alter the arrangement of nucleosomes, allowing the DNA to be more accessible to other proteins in the cell

• using reversible chemical modification of histones that are catalyzed through the different histone-modifying enzymes

  • the tails of the 4 core histones are subject to the covalent modifications which include the additions of acetyl, phosphate or methyl groups

  • these modifications serve as docking sites for regulatory proteins on the histone tails

    • the proteins that attach to these docking sites have varying purposes and lead to different outcomes

heterochromatin

most highly condensed form of interphase chromatin

makes up about 10% of an interphase chromatin

euchromatin: the rest of the interphase chromatin

both euchromatin and heterochromatin are composed of mixtures of different chromatin structures

replication origins:

initiator proteins binding to specific DNA sequences that begins the process of DNA synthesis

the initiator proteins pry the DNA strands apart and break the hydrogen bonds between the bases

simple cells, like bacteria and yeast, are composed of DNA sequences that attract the initiator proteins that are especially easy to open

since A-T base pairs are held together by fewer hydrogen bonds than G-C pairs, A-T pairs are found at replication origins bc they’re easier to break apart

after the initiator protein binds to DNA at the replication origin and opens the double helix

it attracts a groups of proteins that carry out DNA replication called the replication machine

replication machine: cluster of proteins that join together

replication forks

DNA molecules in the process of being replicated that contain Y-shaped junctions

2 forks form at the replication origin

at each fork, a replication machine moves along the DNA to open up the two strands of the double helix and use each strand to make a new daughter strand

each fork moves away from the origin in opposite directions to unzip and copy the DNA as they go (bidirectional in both prokaryotic and eukaryotic chromosomes)

the movement of a replication fork is driven by

the action of the replication machine which consist of the enzyme DNA polymerase

DNA Polymerase catalyzes the addition of nucleotides to the 3ʹ end of a growing DNA strand, using one of the original, parental DNA strands as a template

creates a new strand of DNA that is complementary in nucleotide sequence to the template

polymerization of DNA

involves the creation of a phosphodiester bond between the 3’ end of the growing DNA chain and the 5’ phosphate group of the incoming nucleotide → deoxyribonucleoside triphosphate

at each replication fork

one new DNA strand is being made on a template that runs in one direction while the other is being made on a template in the opposite direction

one new DNA strand is being made on a template that runs in one direction while the other is being made on a template in the opposite direction

makes the replication fork asymmetrical

DNA polymerase adds new subunits only to the 3’ end of a DNA strand → new DNA chains can only be synthesized in a 5’-to-3’ direction (accounts for the leading strand)

for the lagging strand: the DNA strand grows discontinuously in the incorrect 3’ to 5’ direction

okazaki fragments

successive, separate, small DNA pieces

these fragments are later joined together to form a continuous new strand

DNA polymerase can avoid mutations in 2 ways:

• through the monitoring of the base-pairing between each incoming nucleoside triphosphate and the template strand

  • only when the match is correct does the DNA polymerase undergo a small structural rearrangement that allows it to catalyze the nucleotide-addition reaction

• through proofreading

  • used to correct any error made by the DNA polymerase

  • takes place at the same time of DNA synthesis

  • before adding the next nucleotide, the enzyme checks whether the previously added strand is correctly base-paired to the template strand

  • if so, the polymerase adds the next nucleotide

  • if not, the polymerase clips off the mispaired nucleotide and tries again

  • can only proofread from 5’ to 3’ direction so backstitching is required for the lagging strand

To begin the process of DNA polymerase beginning a completely new DNA strand:

primase: enzyme that makes a short length of RNA using the DNA strand as a template

the RNA is then base-paired to the template strand and provides a base-paired 3’ end as a starting point for DNA polymerase

for the leading strand: the RNA primer is needed to start replication at a replication origin; atp, the DNA polymerase takes over by extending the primer with DNA synthesized in the 5’to the 3’ end

for the lagging strand: the movement of the replication fork continually exposes unpaired bases on the lagging-strand template, and new RNA primers are laid down at intervals along the newly exposed, single-stranded stretch

DNA polymerase adds a deoxyribonucleotide to the 3’ end of each new primer to produce another Okazaki fragment and elongate this fragment until it runs into the previously synthesized RNA primer

To produce a continuous new DNA strand from the many separate pieces of nucleic acid made on the lagging strand:

a nuclease removes the RNA primer

a repair polymerase (or DNA Polymerase I) replaces the RNA primers with DNA

DNA ligase joins the 5’-phosphate end of one DNA fragment to the adjacent 3’-hydroxyl end of another

DNA helicases and single-strand DNA-binding proteins:

pry apart the double helix for the incoming nucleoside triphosphates

helicase sits in the very front of the replication machine where it uses the energy from the ATP hydrolysis to propel itself forward

single-strand DNA-binding proteins latch onto the single-stranded DNA which is exposed from the helicase to prevent the strand from re-forming base pairs and keeps them in an elongated form so they serve as templates

DNA topoisomerases

enzymes that relieve the tension on the other side of the double helix as the helicase pries open one side

makes a single-strand nick in the DNA backbone to temporarily release the tension and reseals the nick before falling off the DNA

Sliding clamp

keeps DNA polymerase firmly attached to the template while it is synthesizing new strand of DNA

forms a ring around the newly formed DNA double helix and, by tightly holding the polymerase, allows the enzyme to move along the template strand w/o falling off

clamp loader

replication protein that assembles the clamp around DNA

hydrolyzes ATP each time it locks a sliding clamp around a newly formed DNA double helix

clamp is removed and reattached each time a new Okazaki fragment is made

Telomerase Replicates the Ends of Eukaryotic Chromosomes

lagging strands cannot be replicated all the way to the chromosome tip

when the final RNA primer is removed, there is no enzyme that replaces it with DNA

Bacteria avoid this problem by having circular DNA

Eukaryotes avoid this problem by adding telomeres (which are long, repetitive nucleotide sequences) to the ends of every chromosome

these telomeres attract telomerase (an enzyme that carries its own RNA template) to add multiple copies of the same repetitive DNA sequence to the lagging-strand template

depurination

a spontaneous reaction that removes a purine base from a nucleotide

deamination

spontaneous loss of an amino group from a cytosine in DNA to produce uracil

Basic pathway for repairing damage:

recognize the damaged DNA and remove it (can be done in various ways)

repair DNA polymerase binds to the 3’-hydroxyl end of the cut DNA strand, fills in the gap by making a complementary copy of the info present in the undamaged strand

nick in the sugar-phosphate helix is sealed by DNA ligase

mismatch repair

used to correct errors in DNA strands by recognizing and removing only the newly made DNA

in bacteria: newly synthesized DNA lacks a type of chemical modification that is present in the preexisting parent DNA

when this DNA is unmethylated the new and template strands are easily distinguishable

in humans: mismatch repair plays an important role in preventing cancer

cancers arise from cells that have accumulated multiple mutations, a cell deficient in mismatch repair has a greatly enhanced chance of becoming cancerous

double-strand break:

when both strands of the double helix are damages at the same time

can lead to the fragmentation of chromosomes and the loss of genes

nonhomologous end joining:

hurriedly sticking the broken ends back together before the DNA fragments drift apart and get lost

often loses nucleotides at the site of the repair

homologous recombination

uses the info on the undamaged strands of the intact double helix to repair the complementary strands in the broken DNA with no loss of genetic info

chews back 5’ ends of the 2 broken strands at the break

with the help of specialized enzymes, one of the broken 3’ ends invades the unbroken homologous DNA duplex and searches for the complementary sequence through base-pairing

how does transcription begin

• when a small portion of the DNA double helix is opened to expose the bases on each DNA strand

• one of the two strands serves as a template for the synthesis of RNA

• ribonucleicacids are added to the growing RNA copy chain

  • base-pairing occurs with the DNA template strand

  • when a good match is made, the RNA polymerase covalently bonds the incoming ribonucleoside triphosphate to the RNA strand

  • produces the RNA chain called RNA transcript

transcription vs DNA replication

• RNA strand in transcription doesn’t remain hydrogen bonded to the DNA template strand but the RNA chain is displaced and the DNA helix reforms

• RNA is copied from a small region of DNA

• DNA and RNA polymerases catalyze the formation of the phosphodiester bonds that link the nucleotides together and form the sugar-phosphate backbone of the RNA chain

• RNA polymerase moves along the DNA stepwise, only unwinding the DNA helix just enough to expose a new region for base-pairing

• the rna chain grows one nucleotide at a time in the 5’ to 3’ region

RNA polymerase vs DNA polymerase

• RNA polymerase uses ribonucleoside triphosphates as substrates so the RNA polymerase catalyzes the linkage of ribonucleotides not deoxyribonucleotides

• RNA polymerases can start an RNA chain without a primer and do not accurately proofread their work unlike DNA polymerases

• RNA is not used as the permanent storage form of genetic information in cells

messenger RNA (mRNA)

• RNA encoded by gene and direct the synthesis of proteins

  • typically carries information transcribed from just one gene to code a single protein

  • in bacteria: a set of adjacent genes is often transcribed as a single mRNA which carries info for more than one protein

ribosomal RNA (rRNA):

noncoding RNA that form structural and catalytic core of the ribosomes and translates mRNA into protein

transfer RNA (tRNA):

act as adaptors that select specific amino acids and hold them in place on a ribosome for their incorporation into proteins

micro RNA (miRNA)

serve as key regulators of eukaryotic gene expression

to start transcription (in eukaryotes)

• RNA polymerase must be able to recognize the start of a gene and bind firmly to the DNA at this site

• RNA polymerase collides randomly with a DNA molecule and the enzyme weakly sticks to the double helix and slide along the length of the gene

• when the RNA polymerase latches tightly to the promoter region (which lies immediately upstream from the starting point for RNA synthesis)

• after it binds tightly to this region, the RNA polymerase opens up the double helix in front of the promoter region to expose the nucleotides on each strand of the short stretch of DNA

• one of the two strands acts as a template strand for the complementary base-pairing with the incoming ribonucleoside triphosphates

• elongation continues until the enzyme encounters the terminator regions where polymerase halts and releases both the DNA template and the newly made RNA transcript

• the terminator sequence is also transcribed and the interaction of the 3’ segment of RNA with the polymerase allows the enzyme to let go of the template DNA

to start transcription (in bacteria)

• sigma factor allows for the RNA polymerase to recognize the promoter region on the DNA

• each base has unique features to the outside of the double helix which allows the sigma factor to identify the promoter sequence without having to separate the DNA strands

• when the sigma factor opens the DNA double helix, the sigma factor then binds to the exposed base pairs

RNA polymerases I and III

transcribe the genes encoding transfer RNA, ribosomal RNA, and various other RNAs that play structural and catalytic roles in the cell

RNA polymerase II

transcribes the rest, including all those that encode proteins—which constitutes the majority of genes in eukaryotes

eukaryotic RNA polymerases require the assistance of a large set of accessory proteins to start transcription

general transcription factors, which must assemble at each promoter, along with the polymerase, before transcription can begin

general transcription factors

accessory protein that assemble on the promoter that position the RNA polymerase and pull apart the DNA double helix to expose the template strand

TATA Box:

a short segment of DNA double helix composed primarily of T and A nucleotides, usually located 30 base pairs upstream from the transcription site

development of general transcription factors

• begins with transcription factor TFIID binding to the TATA box

• after binding to the DNA, TFIID causes a dramatic local distortion in the DNA double helix which helps to serve as a landmark for the assembly of other proteins at the promoter.

• after the TFIID binds to the TATA Box, the other factors assemble along with RNA polymerase II to form a complete transcription initiation complex

• once RNA polymerase II has been positioned onto the promoter, it must be released from the complex of the transcription factors to begin marking an RNA molecule

• TFIIH: contains a protein kinase and adds phosphate groups to the tail of the RNA polymerase to liberate it

• once transcription has begun, the general transcription factors dissociate from the DNA

• when RNA polymerase II is done transcribing a gene, it is released form the DNA and the phosphates on its tails are taken off by protein phosphatases

the way the RNA transcripts are handled before translation in bacteria

• since bacteria don’t have a nucleus, their DNA is directly exposed to the cytosol which contains the ribosomes where protein synthesis takes place

• as mRNA molecule in a bacterium starts to be synthesized, the ribosomes in the cytosol immediately attach to the free 5’ end of the RNA transcript and begin translating it into proteins

the way the RNA transcripts are handled before translation in eukaryotes

• DNA is enclosed in the nucleus which is where transcription takes place

• translation occurs on ribosomes in the cytosol

• eukaryotic mRNA are transported out of the nucleus through small pores in the nuclear envelope into the cytosol

• but before it can be exported to the cytosol, the RNA has to go through RNA processing steps which include: capping, splicing and polyadenylation

• the enzymes responsible for RNA processing ride on the phosphorylated tail of eukaryotic RNA polymerase II as it synthesizes an RNA molecule

• RNA capping and polyadenylation occur on all RNA transcripts

  • capping: modifies the 5ʹ end of the RNA transcript

    • Involves an atypical nucleotide (methylated guanine) attached in a unique way.

    • Occurs in eukaryotes after RNA polymerase II synthesizes ~25 nucleotides.

    • In bacteria, the 5ʹ end is simply the first nucleotide of the transcript.

  • Polyadenylation: modifies the 3ʹ end of newly transcribed mRNA.

    • In eukaryotes, the 3ʹ end is first trimmed by an enzyme at a specific nucleotide sequence.

    • A second enzyme adds a series of repeated adenine (A) nucleotides (poly-A tail).

    • The poly-A tail is typically a few hundred nucleotides long.

• both of these modifications increase the stability of a eukaryotic mRNA molecule, facilitate its export from the nucleus to the cytosol and mark the RNA molecule as an mRNA

Introns

Long noncoding sequences that interrupt coding sequences

Exons

Shorter coding sequences that are expressed

Introns Are Removed from Pre-mRNAs by RNA Splicing

• to produce an mRNA in a eukaryotic cell, both introns and exons are transcribed into RNA

• RNA Splicing: Introns are removed, and exons are joined together

  • Splicing can occur before or after the addition of the poly-A tail.

  • Final product: functional mRNA that can exit the nucleus for translation.

  • Prior to splicing, the transcript is called precursor-mRNA (pre-mRNA).

  • carried out largely by RNA molecules rather than proteins

  • small nuclear RNAs (snRNAs), are packaged with additional proteins to form small nuclear ribonucleoproteins (snRNPs - or snurps)

• each intron contains a few short nucleotide sequences that act

  as cues for its removal from the pre-mRNA

• using these nucleotide sequences as a guide, an elaborate splicing machine cuts out the intron in the form of a “lariat” structure

• ribozymes and snRNPs form the core of the spliceosome which is the large assembly of RNA and protein molecules that carries out RNA splicing in the nucleus

• alternative splicing: Eukaryotic gene transcripts can be spliced in various ways.

  • Each splicing variant can produce a distinct protein.

  • Enables the production of multiple proteins from a single gene

anticodon

a set of three consecutive nucleotides that bind through base-pairing to the complementary codon in an mRNA molecule

Aminoacyl-tRNA Synthetases:

Enzymes responsible for recognizing and attaching the correct amino acid to its corresponding 3’ end of tRNA

ribosomes

• Latches onto mRNA.

• Captures and positions the correct tRNAs.

• Covalently links amino acids to form a polypeptide chain.

composition and structure of ribosomes

• Made up of dozens of ribosomal proteins and several ribosomal RNAs (rRNAs).

• Eukaryotic cells contain millions of ribosomes in the cytosol.

• Composed of:

  • Large Subunit: Catalyzes peptide bond formation.

  • Small Subunit: Matches tRNAs to mRNA codons.

• Complete ribosome mass: several million daltons (compared to average protein mass of 30,000 daltons).

Protein Synthesis Process:

• Ribosomal subunits come together on mRNA near its 5ʹ end to initiate protein synthesis.

• mRNA is pulled through the ribosome in a 5ʹ-to-3ʹ direction.

• Ribosome translates the nucleotide sequence into an amino acid sequence, adding each amino acid in the correct order.

• Eukaryotic ribosomes add ~2 amino acids per second; bacterial ribosomes add ~20 amino acids per second.

Ribosome Binding Sites:

• A Site (Aminoacyl site): Charged tRNA enters, base-pairing with mRNA codon.

• P Site (Peptidyl site): Holds the growing peptide chain.

• E Site (Exit site): Spent tRNA is moved before ejection.

Translation Cycle:

• Charged tRNA enters the A site, linking its amino acid to the chain in the P site.

• Large ribosomal subunit shifts, moving spent tRNA to the E site for ejection.

• Cycle repeats with each amino acid addition until a stop codon is encountered, leading to protein release.

Ribozymes

RNA molecules that possess catalytic activity

initiator tRNA:

always carries the amino acid methionine (or formyl methionine in bacteria)

initiation of translation in eukaryotes:

• Small Ribosomal Subunit:

  • Binds to the 5ʹ end of the mRNA marked by the 5ʹ cap.

  • Scans the mRNA in the 5ʹ-to-3ʹ direction to find the first AUG start codon.

• Binding of Initiator tRNA:

  • When the AUG is recognized, initiation factors dissociate.

  • The large ribosomal subunit binds to complete ribosomal assembly.

  • The initiator tRNA is bound to the P site, preparing for the addition of the next charged tRNA to the A site.

initiation of translation in prokaryotes:

• Bacterial mRNAs lack a 5ʹ cap.

• Contain a ribosome-binding sequence (~6 nucleotides) upstream of the AUG start codon.

• Prokaryotic ribosomes can bind directly to start codons within the mRNA, aided by the ribosome-binding site.

• Polycistronic mRNA:

  • Prokaryotic mRNAs often encode multiple proteins, with separate ribosome-binding sites for each coding sequence.

  • Eukaryotic mRNAs typically encode a single protein, relying on the 5ʹ cap for ribosome positioning.

termination of translation in both prokaryotes and eukaryotes:

• signaled by the presence of stop codons in mRNA

• release factors: proteins that bind to any stop codon that reaches the A site on the ribosome

• Alter the activity of peptidyl transferase, causing it to add a water molecule instead of an amino acid.

• This reaction frees the completed polypeptide chain from the tRNA, leading to protein release.

a cell can control the proteins it contains by

• controlling when and how often a given gene is transcribed

• controlling how an RNA transcript is spliced or otherwise processed

• selecting which mRNAs are exported from the nucleus to the cytosol

• regulating how quickly certain mRNA molecules are degraded

• selecting which mRNAs are translated into protein by ribosomes

• regulating how rapidly specific proteins are destroyed after

  they have been made

Transcription Regulators Bind to Regulatory DNA Sequences

• in bacterial and eukaryotic genes, the promoter region of a gene binds to RNA polymerase and correctly orients the enzyme to begin making an RNA copy of the gene

• the promoters include a transcription initiation site where RNA synthesis begins and sequences that contain recognition sites for proteins that associate with RNA polymerase

  • in eukaryotes: general transcription factors

  • in bacteria: sigma factors

• along with the promoter, most genes contains regulatory DNA sequences which are used to switch the gene on or off

  • longer in eukaryotes and act as molecular microprocessors

• to recognize the regulatory DNA sequences, there must be proteins called transcription regulators that bind to the regulatory DNA sequence that act as the switch to control transcription

• many of these transcription regulators bind to the DNA helix as dimers which doubles the area of contact with the DNA which increases the potential strength and specificity of the protein-DNA interaction

Transcription Switches Allow Cells to Respond to Changes in Their Environment

• When tryptophan concentrations are low, the operon is transcribed; the resulting mRNA is translated to produce a full set of biosynthetic enzymes which work to synthesize the amino acid

• When tryptophan is abundant, the amino

  acid is imported into the cell and shuts down production of the enzymes, which are no longer needed

operator

short DNA sequence in the operon’s promoter that is recognized by the transcription regulator

when the regulator binds to the operator

it blocks access of the RNA polymerase to the promoter which prevents transcription of the operon and the production of the tryptophan-synthesizing enzymes

transcriptional repressor:

protein that, when active, switches genes off

transcriptional activator

• proteins that switch genes on and work on promoters that are able to bind and position RNA polymerase on their own

  • often have to interact with a second molecule to be able to bind to DNA

  • ex: the bacterial activator protein CAP has to bind to cyclic AMP (cAMP) before it can bind to DNA

  • genes that are activated by CAP are switched on when there is an increase in concentration of cAMP, which occurs when there is no glucose available, and results in the CAP driving the production of enzymes that allow the bacteria to digest other sugars

lac operon

• is controlled by both the Lac repressor and the CAP activator

• the Lac operon encodes protein required to import and digest lactose

• when there is no glucose, the bacteria make cAMP which activates CAP to switch on the genes that allow the cell to use alternative sources of carbon including lactose

• the Lac repressor shuts off the operon in the absence of lactose which enables the control region of the Lac operon to take in two different signals and meet two conditions: glucose is absent and lactose is present

enhancers

DNA sites to which eukaryotic gene activators bind to enhance the rate of transcription

mediator

additional proteins that serve as adaptors to close the loops