Phys and genetics Maxine topics 1 and 2

The nature of any microbial cell is determined by what?

amount of each protein

which proteins are present

activity of each enzyme(its turnover number)

Protein levels and enzyme activities
are determined:

Genetically, via regulation of gene
expression
Biochemically, via regulation of
enzyme activity

What shape is most bacterial DNA?

Circular

DNA form loops that have varying degrees
of supercoiling.

Plasmid vrs chromosomal dna in bacteria

plasmid genes are not essential for growth/survival

chromosomal has genes essential for growth/survival

Host range and copy number usually determined by what in a plasmid?

ori region

What kind of plasmids can be used for gene cloning?

artificial ones

Can the ori (origin of replication) region be translated?

no

which parts of the chromosomes do not contain genes?

non-coding regions

What is a gene ?

A sequence of nucleotides that usually encodes a protein.
Some encode RNA eg tRNA, rRNA, regulatory RNA

What do proteins do?

Proteins perform most of functions in a cell including catalysis (enzymes), transport,
signal transduction -→ they determine the properties and physiology of cells

What is the central dogma?

DNA→ RNA = transcription

RNA→ Protein =translation

base pairing rules

DNA: A-T, C-G

RNA A-U, C-G

Types of RNA and function

1. messenger RNA (mRNA) – will be translated to proteins
In bacteria, one mRNA transcript may encode several proteins

2. Ribosomal RNA (rRNA) – part of ribosomes
3 sizes: 16S, 23S, 5S

3. Transfer RNA (tRNA) – aminoacylated with amino acids
- decode mRNA sequence to amino acid sequence

4. Small RNA (sRNA) – Regulates transcription or translation

5. Catalytic RNA

What does RNA pol do?

an enzyme that transcribe DNA to RNA, recognizes promoter

What type of protein is associated with RNA polymerase that determine the DNA sequence
that RNA polymerase will bind to initiate transcription?

sigma

Sigma 70 in E. coli is an elongated protein containing how many domains and of what?

containing 4 domains sigma(1,2,3,4)

What is the first step in bacterial transcription initiation?

The RNA polymerase holoenzyme scans DNA for promoter sequences.

What are the two conserved promoter elements the holoenzyme recognizes?

The -35 and -10 sequences (named for their position, in base pairs, upstream of the transcription start site)

What complex forms when the holoenzyme binds the promoter, with DNA still double-stranded?

The closed complex.

What happens to the DNA and enzyme as the open complex forms?

RNA polymerase unwinds the DNA (forming the transcription bubble) and begins synthesizing RNA using rNTPs.

What byproduct is released when rNTPs are added to the growing RNA chain?

Pyrophosphate (PPi)

What is the region of locally unwound DNA in the open complex called?

The transcription bubble.

What is the final step once RNA synthesis is underway?

The sigma factor leaves the complex, leaving only the core enzyme to continue transcription.

RNA polymerase binding to the promoter bends the DNA, causing melting (strand separation). Which sigma factor regions contact the -10 and -35 sequences?

σ2 contacts the -10 sequence, and σ4 contacts the -35 sequence.

5’ untranslated region in mRNA contains what?

Shine-Dalgarno sequence

What and where is the +1 position on DNA?

The transcription start site the first nucleotide of DNA that gets transcribed into mRNA.

downstream -35 and -10

What is a strong promoter?

RNA pol binds close to consensus seq

what is a weak promoter?

RNA pol binds far away from consensus seq

RNA base sequence is very similar to the sequence of the which strand?

coding strand except T is replaced with U in the RNA

Is the signal for transcription termination on the DNA or RNA ?

The RNA (mRNA)

What two RNA sequence features together signal Rho-independent termination?

A GC-rich stem-loop (hairpin) structure followed immediately by a run of U residues (poly-U).

In Rho-independent termination what happens to RNA polymerase when the stem-loop and NusA make contact with it?

RNA polymerase pauses.

In Rho-independent termination why does the RNA-DNA hybrid separate (melt) at the poly-U/poly-A region during termination?

Because A-U base pairs are weak (2 hydrogen bonds not 3 like C-G), making them easy to melt once RNA polymerase pauses.

In Rho-independent termination what protein makes contact with RNA polymerase (along with the hairpin) to cause termination?

Nus-A

What accessory protein is required for Rho-dependent termination?

Rho

What type of sequence does Rho bind to on the mRNA?

C-rich regions (specifically the rut site)

What structural form does Rho take when the RNA is threaded through it?

A hexamer (six-subunit ring structure).

How does Rho ultimately cause transcription termination?

Through ATP-dependent helicase activity that separates the RNA-DNA hybrid duplex.

What event directly causes termination once Rho reaches RNA polymerase?

Contact between Rho and RNA polymerase.

What is the significance of the "pause site" on RNA polymerase?

It's where RNA polymerase slows down, giving Rho time to catch up along the RNA before triggering termination.

A Ribosome (70s) consists of what subunits?

small subunit (30s)

large subunit (50s)

The genetic code is a sequence of 3 base called what?

codons

Stop codons do what?

signal the ribosome to stop translation and dissociate from the mRNA template

What is the job of tRNA?

Read the codon on the mRNA and translate the codon to a particular amino acids

tRNA anticodon base pair with what?

mRNA codon

An amino acid is covalently attached to which
end of the tRNA?

3’

Specific tRNAs are charged with specific amino acids by what?

amino acyl tRNA synthetases.

5’ untranslated region in mRNA is and has what?

the leader sequence
and contains the Shine-Dalgarno sequence after +1

What ribosomal component base-pairs with the Shine-Dalgarno sequence on the mRNA?

The 3' end of the 16S rRNA.

What is another name for the Shine-Dalgarno sequence?

Ribosome binding site (RBS).

What is the first codon (initiation codon) used to start translation?

AUG

Although AUG normally encodes methionine, what is the first amino acid actually incorporated in most bacteria?

Formyl-methionine (fMet).

What does the ribosome slide down the mRNA to find after binding the Shine-Dalgarno sequence?

The start codon (usually AUG)

What special amino acid does the initiator tRNA carry when it base-pairs with the AUG start codon?

Formyl-methionine.

What is the three-nucleotide sequence on tRNA called that base-pairs with the mRNA codon?

Anticodon.

What type of bond forms between the growing polypeptide and the newly added amino acid?

peptide bond

Polycistronic versus monocistronic mRNA

polycistronic mRNA encodes multiple genes

monocistronic mRNA encodes one gene

What is an operon?

group of genes that are transcribed together into a single polycistronic mRNA, under control of a shared promoter

Are Transcription and translation coupled?

yes meaning they occur at the same time!

possible bc transcription is in nucleus and translation in cytoplasm

What happens to the protein in a missense point mutation?

A single amino acid is changed to a different amino acid bc one base pair changes

What happens in a nonsense point mutation?

A premature stop codon is introduced,ending the protein early bc of one base pair change.

What is the term for a mutation caused by insertion or deletion of nucleotides that shifts how mRNA is read in codons?

Frameshift mutation.

Which type of mutation would generally have the most drastic effect on protein function?

Frameshift mutations (insertions/deletions not in multiples of 3).

What is a mutation called when it doesn't change the amino acid sequence of the encoded protein?

A silent mutation.

What physically happens to the DNA in an inversion mutation?

A segment of DNA is reversed/flipped in orientation.

How can mutations arise?

Spontaneously, or from exposure to mutagens (e.g., UV light, nitrous acid).

Bacteria genome such as in E. coli has how many ORFs?

4000

Are all of the encoded proteins produced in the cell all the time?

No would take too much Energy each AA takes up 4ATP

How can bacteria regulate their gene expression ?

under certain conditions

constitutively expressed genes are what?

genes that are always expressed in the bacteria normally
essential for cell survival. Some examples includes DNA polymerases, RNA polymerase
etc.

How can RNA transcript or protein levels be regulated in the cell?

Multiple possible points of control:

1) Regulation at the transcription initiation step
- by repressors or activators

2)Regulation at the transcription elongation step (attenuation)
- RNA polymerase can terminate transcription prematurely resulting in the
synthesis of a short mRNA that normally does not encode any genes.

Whether the RNA polymerase will terminate transcription prematurely can be
regulated by what?

Either ribosomes or proteins called antiterminators

Repressors do what?

Prevent access of RNA polymerase to promoters by binding to DNA close to
the promoters and either

1. physically blocks access of RNA polymerase

2. Bend DNA to prevent binding of RNA polymeras

Activators help recruit RNA polymerase to promoters of genes by?

1. binding DNA upstream of promoters and physically interacting with components of
RNA polymerases
2. Activators can also enhance transcription by catalyzing the formation of the open DNA
complex

How can u make an activator a repressor?

changing DNA binding site:

By moving its operator (DNA binding site) so that it overlaps the promoter (-35/-10 region) or the RNA polymerase binding footprint, instead of positioning it upstream where it can cooperatively contact RNA polymerase.

An operator is a region in DNA where?

1) Activator and Repressors bind

location is important to transcription

How does a repressor bind DNA when gene expression is controlled by induction?

he repressor binds DNA directly, without needing a ligand — it represses the gene by default.

What type of ligand causes a repressor to release from DNA in induction?

An inducer

What is the process called where ligand binding turns gene expression ON by causing a repressor to release from DNA?

Induction

What must happen before a repressor protein can bind DNA in derepression-controlled genes?

The repressor must first bind a corepressor ligand, forming an active repressor-corepressor complex.

What is the process called where the absence of a corepressor allows gene expression to turn ON?

Derepression

What is the key functional difference between induction and derepression in terms of the repressor's default state?

In induction, the repressor is active by default (bound to DNA) and is inactivated by an inducer. In derepression, the repressor is inactive by default (can't bind DNA alone) and is activated by a corepressor.

How does the activator protein turn on transcription of the target gene?

It first binds a ligand (inducer); the resulting complex binds the regulatory sequence and activates the gene

What happens to the target gene once the activator-inducer complex binds the regulatory sequence?

The target gene is expressed — RNA polymerase transcribes it into mRNA.

What happens when the ligand (inducer) is removed from the activator?

Transcription stops, and the target gene is no longer expressed.

Factors that influence gene transcription can be divided into?

1)Cis elements

2)Trans elements

What is a cis element?

DNA regions encoded in a plasmid or in a chromosome in the vicinity of a gene. eg promoter, operator

What is a trans element?

All the diffusible cellular molecules that are able to bind
to the DNA. eg. Transcription factors (activators or repressors)

What are Transcription factors?

Diffusible molecule (usually proteins) that binds upstream of genes
to regulate their level of transcription

Transcription factors can regulate what?

1) specific genes or operons

2)number of genes/ operons global regulators

Transcription factors are classified into families by what?

based on AA seq simularity

Many Transcription factors have what motif for DNA binding?

helix-turn-helix

Helix binds to the major groove of DNA double helix allowing amino acids in helix to
interacts with specific bases of DNA

Nomenclature differntiation between gene and protein?

1. Genes are normally written as a three letter code followed by a Letter. The three letter
code are in small letters and the Letter is capitalized. All are italicised.
lacZ, trpA

2. Proteins are also written in three letter code followed by a letter but the first letter
of the three letter code is capitalised. Letters should not be italicised.
eg. LacZ, Trp

Lactose is made of which monosaccarides?

Glucose and galactose

What protein transports lactose from outside the cell to inside the cell?

Lactose permease (LacY)

What enzyme converts lactose to allolactose (at low levels) and breaks lactose into glucose and galactose (at high levels)?

β-galactosidase (LacZ)

What product forms when low levels of β-galactosidase act on lactose, and goes on to induce the lac operon?

Allolactose

When high levels of β-galactosidase act on lactose, what two products are formed?

Galactose and glucose

What is the functional role of allolactose in regulating the lac operon?

It binds the lac repressor, causing it to release from the operator so transcription can proceed

What does lacZ encode?

β-galactosidase, which hydrolyzes lactose into galactose and glucose.