Biochem Test 2

Central Dogma

The one way flow of genetic information: DNA → RNA → Protein. It cannot go backwards

DNA is a polymer of nucleotides linked by?

phosphodiester bonds. directionally from the 5’ phosphare to the 3’ hydroxyl end.

How is the double Helixx stablized

hydrogen bonds and hydrophobic stacking.

Chargaffs rules

adenine = thymine

guanine = cytosine

total purines ( A + G) = total pyrimidines (C + T)

How many bonds does Adenine form with its pair, how about guanine?

A 2 bonds w/ T

G 3 bonds W/ C

Are Dna strands parrallel or antiparallel

antiparallel

The backbone of DNA is formed by

repeating deoxyribose-phosphate units linked by phosphidester bonds

How do the major and minor grooves of DNA allow for sequence-specific recognition?

By exposing unique hydrogen-bond donor and acceptor patterns for each base pair.

Which amino acids make the largest number of hydrogen bonds?

Arg, Lys, Ser, Thr, Asn, and Gln

what is the negative aspect of intercalating agents

many are mutagenic and carcinogenic

How do intercalating agents affect DNA

They insert between base pairs, distorting DNA and potentially causing replication/ transcription blockage, mutations, and cancer

How is DNA replication initiated in bacteria

DnaA binds at the AT- rich oriC, unwinding DNA to form two replication forks for bi direction, semi conservative replication

Can DNA polymerase add nucleotides without and existing nucleic acid?

No, therefore it needs a primer

What direction does DNA polymerase synthesize DNA

5’ → 3’

explain how DNA replication in prokaryotes is semi-discontinuoys

The leading strand is synthesized towards the fork while the lagging strand is synthesized discontinuously away from the fork

How does the cell determine where DNA replication begin in bacteria

• oriC: AT-rich origin of replication recognized by DnaA protein.

• Unwinding: Creates an open replication bubble with two replication forks.

• Replication: Proceeds bidirectionally and semi-conservatively.

after prokaryotic DNA has been unwinded what binds to the single strands and why

SSB proteins bind to the single strands to keep them separated and protected, allowing replication to proceed

What does DNA polymerase do?

Remove RNA primers (5’ → 3’ exonuclease) and fills gaps

What proofreads DNA and how

DNA polymerase III 3’ → 5’ (exonuclease activity)

removes incorrectly incorporated nucleotides from the 3’ end of the growing strand

When does proofreading occur

immediately after a misincorporation before synthesis continues

What is the speed of DNA synthesis by DNA polymerase III

~749 nucleotides per second

How do Type 1 DNA topiomerases work

cut one DNA strand

pass the other strand through the break

reseal the nick

How do Type II DNA topoisomerases work

Cut both strands

pas another DNA segment through the break

reseal the break

Why are DNA topoisomerases important

these enzymes relieve supercoils and are essential for DNA replication and transcription

Whats different about Eukaryotic DNA replaction than Prokaryotic

Eukayotic needs for replication:

multiple origins

multiple polymerases

multiple enzymes

Differences of pro and euk in assembly of DNA into chromosomes

Pro is naked (minimal proteins)

euk is tightly packed with histones into chromatin forming nucleosones

What are many effective antiviral and anticancer drugs and what do they target

nucleoside analogs

target the 3’ hydroxyl group which blocks DNA synthesis (causes chain termination/ inhibits polymerase activity)

What does Reverse transcriptase do?

Enables retrovirus to convert RNA to DNA.

When do mismatches mutation arise

during DNA replication when incorrect nucleotides are incorporated.

What is a transition mutation? is it more or less common than transverse mutations?

they replace a purine with a purine (A ←→G) or pyrimidine with a pyrimidine (C←→ T)

more common

what are the cell cycle checkpoints and what do they do

G1 and G2 prevent the propagation of damaged DNA by halting cell cycle progression until damage is repaired

Thymine dimers and double strand breaks are major forms of?

DNA damage, if not repaired they can cause mutations, genomic instability and cancer

How does direct reversal repair work

restores the original base by reversing the chemical modification without using a template and without cleaving the DNA backbone

How can defects in DNA repair pathways affect cancer risk?

Defective DNA repair causes mutations to accumulate, increasing the risk of specific types of cancer depending on the pathway affected.

What are Vinyl chloride and styrene? What do they do

procarcinogens

DNA damage by alkylation

What about intercalating agents anatomy allows them to unwind and lengthen the DNA helix

flat, hydrophobic

allows them to slip between base pairs

What non-cancerous cells are also affected by chemotherapeutic agents

bone marrow, gut epithelium, skin/hair follicles (highly proliferative normal tissues)

How do chemotherapeutic agents work

by targeting DNA synthesis by inhibiting enzymes or damaging DNA selectively killing rapidly diving cancer cells

Ames Test

A significant increase in revertant colonies indicates that the test compound is mutagenic and potentially carcinogenic

What are oncogenes

cancer causing genes

what happens to Proto oncogenes that face alterations that increase their activity/expression

convert into oncogenes

driving uncontrolled cell proliferation and cancer

How is RNA synthesis catalyzed

by RNA polymerase,

in the 5’ → 3’ direction

What do RNA polymerase require?

DNA template, NTPs. and divalent metal ions

Which enzyme intiated transcription at promoters

sigma + core

Major phases of RNA transcription

initiation, elongation, and termination

How does the sigma subunit confer promoter specificity to RNA polymerase

by binding promoter sequences. promoting open complex formation, and then dissociating after initiation to recycle

RNA polymerase unwinds DNA ahead of it

maintains an ~8-9 bp RNA-DNA hybrid

What are the two termination steps of RNA synthesis

intrinsic termination

rho-dependent termination

Why is eukaryotic transcription more complex than in prokaryotes

chromatin structure, transcription factors, multiple RNA polymerases, distant enhancers. RNA processing events

How do basal transcription factors help in transcription?

help recruit and activate RNA polymerase II at the promoter to initiate transcription

What are enhancers

cis acting elements that do not initiate transcription on their own but stimulate transcription of target genes by recruiting transcription factors and promoting interaction with the promoter

What does posttranscriptional processing do

removes nonfunction spacer sequences from the primary transcript and produces mature functional rRNAs essential for ribosome assembly

What does RNA polymerase III do

transcribes tRNA genes to produce precursor tRNAs that are processed into mature tRNAs

What are ribozymes

RNA catalysts capable of precise RNA cleavage and ligation. (promising as therapeutic agents agaonst cancer, viral infections)

Steps of posttranscriptional modification

5’ capping, 3’ polyadenylation ( addition of poly-A tail), removal of intron that is exported to the cytoplasm

what percentage of inherited genetic diseases result from mutations that disrupt pre-mRNA splicing

15%

How do splice-site mutations cause beta-thalassemia?

Disrupt mRNA splicing, producing abnormal or unstable transcripts and reducing functional beta-globin.

Differences between siRNA and miRNA

si: starts from dsRNA; perfect pairing

mi: starts from endogenous hairpin; partial pairing

What does the mRNA degradation do

ensures proper gene expression control and removal on unneeded transcripts

what do rifampin, actinomycin D, and alpha-amanitin do

inhibit RNA synthesis at different stages/mechanisms

missense mutation

changes one amino acid

nonsense mutation

converts a codon for an amino acid into a stop codon, producing truncated protein

silent mutation

changes a codon without altering the amino acid

Coding mutations: addition/deletion of a base

frameshift downstream

wobble hypothesis

one tRNA can recognize multiple synonymous codons because pairing at the third codon position is flexible

what is Aminoacyl-tRNAs synthetase role in protein synthesis

uses ATP to activate amino acids and attacth the correct amino acid to its matching tRNA, making charged tRNA

How many nucleotides does bacteria initiation begin with

25 nucleotides from 5’ end of the mRNA

shine-dalgarno sequence

sequences in an untranslated region between 5’ end and the first codon translated

What does elongation require

the cleavage of two high-energy bonds per amino acid addition. one while charging tRNA one during translocation

What happends to proteins after synthesis

they are delivierd to

1. internal sites such as mitochondria and nucleus

2.the endoplasmic reticulum for protein targeting/sorting

How are proteins directed to their correct location

targeting information within the protein sequence (peptides and transmembrane)

Histone code hypothesis

proposes that specific combinations of modifications. as well as the order in which they occur help determine chromatin configuration and influence transcription

how do antibiotic and toxins inhibit protein synthesis

act at distinct steps of protein synthesis blocking translation and preventing production

what are the two things that assemble the transcription machinery and determine gene expression

cis elements: DNA sequences

Trans-factors: proteins that bind cis-elements and regulate transcription

What regulates gene expression

lipid soluble hormones

water soluble hormones

How do lipid soluble hormones work

Bind intracellular receptors that act as transcription factors, regulating gene expression at hormone response elements on DNA. (can cross cell membrane)

How do water soluble hormones work

Bind to cell-surface receptors, activating second messengers and protein kinases that alter gene expression and cellular responses. (NOT cross cell membrane)

Why does alternative splicing play an important role in biology

greatly expands the coding potential of the human genome, generating multiple protein isoforms w/ distinct functions

post-transcriptional RNA editing

expands protein diversity by altering nucleotide sequences after transcription; creating different proteins from the same gene

What happens when intracellular iron is low

IRPs stabilize TfR mRNA and inhibit ferritin mRNA so that cells increase uptake and reduce storage

what does 3’ iron response element (IRE) do

IRP binding stabilizes mRNA → increases transferrin receptor → increase iron uptake

what does 5’ IRE do

IRP binding inhibits translation → decreases ferritin synthesis → reduces iron storage

How is complementary DNA duplex created

from mRNA by using reverse transcriptase to synthesize

two facts about cDNA

1.does not contain introns

2.lacks the regulatory regions

Gel electrophoresis

separates DNA fragments by size

an essential technique for analyzing PCR products, and restriction digests

Southern blot is for

DNA

northern blot is for

RNA

Western blot is for

proteins

uses antibody-antigen interactions

what is qRT-PCR

sensitive, quant. method used to measure gene expression (mRNA) in real time

steps of real time PCR

reverse transcription → PCR amplification → flurescene detection → data analysis

SDS

denatures proteins and imparts a uniform negative charge, reduces disulfide bonds, separates proteins based on size

Enzyme linked immunosorbent assay (ELISA)

used for detecting and measuring proteins in biological samples

What is a promoter

a DNA sequence where proteins bind to start transcription

What is transcription

the process of copying DNA to RNA

What is an operator

short DNA segment in an operon that acts as an on/off switch by binding repressor proteins to control whether nearby genes are transcribed

What is an operon

functional unit of DNA in prokaryotes where multiple related genes are clustered under a single promoter and transcribed together into one mRNA molecule

What is the function of the lac operon

regulated genes that allow E. coli to use lactose

What happens to the lac operon when lactose is absent

the active repressor binds the operator, blocking transcription

What happens in the lac operon when lactose is present

lactose forms allolactose, which binds and inactivates the repressor

What is the function of allolactose

binds the repressor, preventing it from binding the operator