Topic 10 - Bacterial Transcription

why are death cap mushrooms so deadly?

because they produce a toxin called a-Amanitin which inhibits RNA polymerase II → stops mRNA production, halts protein synthesis

Genes as a set of developmental switches

all cells have the same set of genes, but in each cell, a different subset of genes are activated and inactivated

There is no need to invent new genes to make grand changes in the body plan of an animal. All you need to do is

switch the existing genes on and off in different patterns

to what degree does subtle changes in expression of developmental genes affect phenotypes

can dramatically alter them → macroevolution(large-scale evolutionary changes, such as the development of new body plans or major differences between species)

“Old Genes, New Regulation”

idea that evolution can create new traits, body structures, or species differences without needing to invent new genes. Instead, changes in how existing genes are regulated (when, where, and how much they are turned on or off) can drive major evolutionary transformations

• Genes stay the same but expression changes due to mutations in regulatory DNA(enhancers, silencers, and promoters)

• small tweaks in regulatory sequences → can lead to big phenotypic changes without having to alter the actual coding of the genes

Human vs. Chimp Evolution

both species share a very similar genome but key differences arise from changes in a small number of regulatory sequences that affect gene expression → large phenotypic difference

• network of interacting genes encoding transcription factors that are differently expressed in human and chimpanzee brains

• example of “Old genes, New Regulation” in Evolution

Transcription Factor

proteins that regulate other genes by turning them on or off

Human brain vs. ape brain

human brain larger than ape brain due to a difference in the timing of gene expression

• precursor cell changes shape later in humans compared to apes which is controlled by transcription factor ZEB2

limb elongation

expression differences in a single gene give rise to limb elongation

adaptive radiation

occurs when a single ancestral species rapidly diversifies into multiple new species, each adapted to different environments or ways of life

Galapagos finches

15 species of finches have evolved from a common ancestor(500,00 years ago) and each of these species adapted to different diets and habitats

natural selection is not constant over - and can result in rapid —

time; evolutionary change

nature selection→ evolution example

drought caused more hard-woody seeds which favored larger beaked birds

General emerging picture

Gene expression changes drive adaptive radiation. What makes all the difference is how much you turn a gene on, when you turn it on, when you turn it off.

single strand of RNA vs. single strand of DNA in terms if chemicals it is made from

RNA uses ribose in place of deoxyribose and uracil in place of thymine

mRNA (messenger RNA) transcript

portable gene that is smaller and more mobile than the DNA sequence, but contains the same information transciprio

transcription

synthesis of an RNA molecule from a DNA template

transcription requires

• A DNA template (the 3’-5’ (anti?)sense strand)

• the transcription unit

  • promoter

  • RNA-coding sequence

  • Terminator

• the raw materials (ribonucleotide triphosphates) (NTPs) needed to build a new RNA molecule

• The transcription apparatus, consisting of the proteins necessary for catalyzing the synthesis of RNA

transcription unit

region of DNA that is transcribed into RNA

• promoter

• RNA-coding sequence

• Terminator

promoter

A DNA sequence where RNA polymerase binds to start transcription

RNA coding sequence

actual part of DNA that is copied into mRNA

terminator

a sequence signaling RNA polymerase to stop transcription

ribonucleotide triphosphates (NTPs)

Uracil (U); adenine (A); guanine (G); cytosine (C)

transcription apparatus

RNA polymerase + proteins needed to build mRNA

RNA polymerase in transcription

binds to the promoter, reads the DNA template, and builds a complementary mRNA strand

Transcription consists of 3 stages

Initiation, Elongation, and Termination

DNA binding proteins and parts

proteins that interact with DNA to regulate gene expression; domains and motif

domains

• part of DNA binding protein

• 60-90 amino acids

• responsible for binding to DNA by forming hydrogen bonds with DNA

motif

• Within the binding domain

• simple structure that fits into the major groove of the DNA and usually recognizes a specific DNA sequence

  • Distinctive types of DNA-binding proteins based on the motif

how does transcription begin

when RNA polymerase binds to a promoter sequence near the beginning of a gene (directly or through helper proteins)

initiation site

site on the DNA from which the first RNA nucleotide is transcribed

• aka: +1 site

upstream

nucleotides that come before the initiation site and are given negative numbers

downstream

nucleotides that come after the initiation site and are marked with positive numbers

consensus sequences

• short stretches of DNA sequences that consist of the most commonly encountered bases at each position in a group of related sequences

• guide RNA polymerase to the correct place to start in transcription

Y indicates what in consensus sequence

pyrimidines

R indicates what in consensus sequence

Purines

N indicates what in consensus sequence

no particular base is more common

C/G indicates what in consensus sequence

cytosine and guanine are equally common

RNA polymerase enzyme size and subunits in organisms even simple ones

large enzymes with multiple subunits

Bacterial promoter has how many major consensus sequences and what are they

two

• 10 consensus sequence (Pribnow box)

• 35 Consensus Sequence

10 Consensus sequence

• located 10 bp upstream of the start site

• Pribnow box (similar to TATA box in eukaryotes)

  • 5’ TATAAT 3’

  • 3’ ATATTA 5’

35 Consensus sequence

• location: 35 bp upstream of start site

• TTGACA

How many subunits does the core enzyme in bacterial RNA polymerase consist of?

5 sub units

sigma factor (σ)

protein that binds to the core enzyme of bacterial RNA polymerase and forms the holoenzyme

holoenzyme

• formed when the sigma factor binds to the core enzyme (bacterial RNA polymerase)

• capable of binding to a promoter and initiating transcription

RNA polymerase of bacteria must bind to a sigma factor to initiate transcription. True or False

True

What strand is transcribed to form RNA

ONLY the sense (template) strand

initiation: first phase of transcription (bacteria)

• RNA polymerase holoenzyme attaches to the promoter

• formation of a appx 20 bp transcription bubble and loss of sigma factor

• RNA polymerase moves downstream from the promoter and RNA synthesis begins

• no primer is needed

elongation: second phase of transcription (bacteria)

• RNA polymerase “moves” downstream

• RNA is synthesized 5’ to 3’

• RNA transcript is nearly identical to the non-template strand of DNA except that uracil replaces thymine and sugar is ribose

elongation (steps)

1. RNA synthesis is complementary and antiparallel to the template strand

2. New nucleotides are added to the 3’- OH group of the growing RNA; so transcription proceeds in a 5’→3’ direction

3. non template strand is not transcribed

what way is RNA synthesized

5’ to 3’

Which strand(s) are transcribed?

template strand; non template strand is not usually transcribed

what is termination and when does it occur

process of ending transcription and it only happens once the polymerase transcribes a sequence of DNA known as the terminator

Rho-independent terminator contains (bacteria)

an inverted repeat followed by a string of approximately six adenine nucleotides

rho-independent termination

one of the mechanisms used by bacteria to terminate transcription

• does not require the Rho protein for termination

• transcription process ends bc the RNA forms a hairpin structure (formed by inverted repeats rich in C and G, followed by a string of uracils), and the weak A-U base pairs cause the RNA polymerase to detach from the DNA, ending transcription

rho-independent termination process (steps)

1. Rho-independent terminator contains an inverted repeat followed by a string of approximately six adenine nucleotides

2. The inverted repeats are transcribed into RNA (2 inverted repeats)

3. The string of U’s (poly-U tail (chat-gpt)) causes the RNA polymerase to pause…

4. …and the inverted repeats in RNA fold into a hairpin (stem-loop structure)…

5. …which destabilizes the DNA-RNA pairing

6. The RNA transcript separates from the template, terminating transcription

Rho dependent termination

transcription termination mechanism in bacteria that requires the Rho helicase to stop transcription

• the Rho protein binds to the rut site on the mRNA, follows RNA polymerase, and uses helicase activity to unwind the RNA from the DNA → termination

Rho-dependent transcription process (steps)

1. Rho binds to the rut site and moves toward the 3’ end

2. When RNA polymerase encounters a terminator sequence, it pauses and rho catches up

3. Using helicase activity, who unwinds the DNA-RNA hybrid and brings an end to transcription

function of sigma factor?

sigma factor controls the binding of RNA polymerase to the promoter

How do amino acids in DNA-binding proteins interact with DNA?

a. By forming covalent bonds with DNA base

b. By forming hydrogen bonds with DNA base

c. By forming covalent bonds with sugars

b.

What binds to the -10 consensus sequence found in most bacterial promoters?

a. The holoenzyme (core enzyme + sigma factor)

b. The sigma factor alone

c. The core enzyme alone

d. mRNA

a.