UF BCH4024 Exam 4: Prokaryotic Transcription

define messenger RNA (mRNA) and describe its function:

contains sequence of bases that encode the primary amino acid sequence for a protein; serves as the template for ribosomal translation

define transfer RNA (tRNA) and describe its function:

carries an amino acid to the catalytic site of a ribosome; base pairs to mRNA to ensure correct incorporation of amino acids into a nascent polypeptide chain

define ribosomal RNA (rRNA) and describe its function:

structural components of a ribosome, where translation occurs

define noncoding RNA (ncRNA) and describe its function:

don't encode proteins, but have a variety of catalytic, structural, & regulatory functions

what are the 2 components comprising genes?

the DNA encoding the protein & DNA regulatory elements needed for transcription

what is the name of the process that uses genes to create an RNA primary transcript?

transcription

how does transcription differ between eukaryotes & bacteria?

in bacteria, the primary transcript is mRNA & is ready for translation, in eukaryotes, the primary transcript has to be modified into final mRNA before it is ready for translation

what are three components of code are present in all genes?

1. promotor

2. open reading frame

3. terminator

in mRNA, if the open reading frame codes for a macromolecule (e.g. protein or ncRNA) the open reading frame can also be called a:

cistron

what is an operon?

a cluster of coordinated genes (similar functions) within prokaryotes that have multiple open reading frames (ORFs) with related functions & are transcribed together into one RNA molecule

how are ORF encoding proteins arranged in a transcription unit?

from 5'→3'

in a prokaryote, use of one promotor & one terminator with multiple ORFs that each encode for a different protein are called:

polycistronic mRNA

in both prokaryotes & eukaryotes, the regulation of which genes are being expressed comes from the combination of which two components?

trans-acting factors & cis-acting elements

describe trans-acting factors:

diffusible (don't have fixed place in genome) factors that can function at multiple sites in a genome & bind to parts of DNA to regulate the degree of transcription expression (i.e. transcription factors)

what are transcription factors?

DNA-binding proteins that determine whether transcription will occur

describe cis-acting elements:

have a fixed place in the genome & only affect the gene they're associated with (typically a DNA sequence)

while DNA replication replicates the entire chromosome/genome, RNA synthesis:

is much more selective

where is the transcription start site of a gene always located?

+1

what enzyme catalyzes transcription?

RNA polymerase

where DNA synthesis requires an RNA primer, RNA polymerase can start synthesizing RNA ____ __________

de novo

what is the substrate of RNA polymerase? what kind of bond does it form to initiate RNA synthesis?

ribonucleoside 5'triphosphate (NTP) is the substrate & RNA polymerase forms a phosphodiester bond between 2 NTPs

the 5' end of an RNA molecule will contain:

3 phosphate groups

do RNA polymerases have proofreading exonuclease activity like DNA polymerases?

no, so errors are more common in RNA synthesis than DNA synthesis

2 polynucleotides can only base pair under what two conditions?

1. their sequences are complimentary

2. they can base pair in an antiparallel fashion

prokaryotes only have one RNA polymerase but two versions of it- what are they?

RNA polymerase core enzyme & RNA polymerase holoenzyme

what are the subunits comprising the RNA polymerase core enzyme involved in prokaryotic transcription?

α₂ββ'ω

which version of RNA polymerase is responsible for the majority of RNA synthesis?

RNA polymerase core enzyme

what is the role of the α subunits of the prokaryotic RNA polymerase core enzyme?

they're essential for enzyme assembly & interact with activators

what is the role of the β & β' subunits of the prokaryotic RNA polymerase core enzyme?

β & β' come together to form the catalytic core/site

what is the role of the ω subunit of the prokaryotic RNA polymerase core enzyme?

structural stability

what is the prokaryotic RNA polymerase holoenzyme responsible for?

transcription initiation & synthesis of the first 10 base pairs

what is the accessory subunit of the prokaryotic RNA polymerase holoenzyme & what is its function(s)?

the σ subunit recognizes promoters & initiates transcription

what qualities are unique to the σ subunit of the RNA polymerase holoenzyme?

it can read DNA, identify promotor sequences, & bind to them; without the σ subunit, the polymerase core enzyme cannot locate promoters, therefore, transcription will not occur

the majority of E. coli promotors are recognized by which σ holoenzyme subunit?

σ⁷⁰

what are the 3 main stages of transcription?

initiation, elongation, & termination

which version of the RNA polymerase is involved in the initiation step of transcription?

RNA polymerase holoenzyme

which version of the RNA polymerase is involved in the elongation step of transcription?

RNA polymerase core enzyme

which version of the RNA polymerase is involved in the termination step of transcription?

RNA polymerase core enzyme

in which direction does the RNA polymerase core enzyme move along the template strand in the elongation step? in which direction does it add rNTPs to the growing RNA strand?

moves along the template strand from 3'→5'

synthesizes RNA from 5'→3'

which rate in prokaryotic transcription is the major determinant of gene expression?

the rate of transcription initiation

if the σ subunit binds to the top strand, which strand will be the template strand?

the bottom strand

if the σ subunit binds to the bottom strand, which strand will be the coding strand?

the bottom strand

if the σ subunit binds to the top strand, which strand will be the coding strand?

the top strand

what is the consensus sequence of the coding strand recognized by σ⁷⁰ at the -10 region?

(5') TATAAT (3')

what is the consensus sequence of the coding strand recognized by σ⁷⁰ at the -35 region?

(5') TTGACA (3')

what are the two bacterial promoter sequence regions recognized by σ⁷⁰?

-35 & -10

what is the UP (upstream promoter) element?

some prokaryotes have an additional AT-rich sequence region that is upstream of the promotor & can interact with the α subunit of RNA polymerase; typically UP elements increase the amount of gene transcription because by binding to the α subunit, we are increasing the number of interactions between the holoenzyme & promoter region

what are constitutive promoters?

promoters that are always active & available for RNA polymerase to transcribe

most of the time the genes that have constitutive promoters are called:

housekeeping genes; required for constant cell maintenance so always available for transcription

constitutive promoters have a variety of ___________ levels. why?

expression because there will be differences in their sequences that will affect the transcription rate

how is a strong promoter defined?

they have very high sequence similarity with the promoter consensus sequence; produce lots of transcripts

how is a weak promoter defined?

they have several base differences; low level of transcription since RNA polymerase has a low affinity for them

how is the rate of transcription initiation determined?

by similarity to the bacterial promoter consensus sequence

what are inducible promoters?

genes that can be turned on & off in response to environmental changes

what is the most regulated step in determining whether a gene is expressed?

rate of transcription initiation

what do specificity factors do?

alter the specificity of RNA polymerase for a given promoter or set of promoters

what do repressors do?

restrict access of RNA polymerase to the promoter

what do activators do?

enhance the RNA polymerase-promoter interaction

what is the specificity factor we discussed in lecture? how does it function as a specificity factor?

σ, its interaction with the promoter will affect the transcriptional rate

what three molecules can influence the transcriptional rate of inducible promoters?

1. specificity factors

2. repressors

3. activators

what structural feature of some proteins allow them to bind DNA? describe:

DNA-binding domains; possess one or more identifiable structural motifs that allows for sequence-specific binding to DNA

what is the predominate DNA binding structure in E. coli?

helix-turn-helix motif

in the helix-turn-helix motif, which component of the structure is responsible for making sequence-specific interactions? why?

recognition α-helix:

- α-helix sticks out from the protein

- interacts with base pairs through major or minor groove

- forms H-bonds & van der Waals with base pairs

what do additional non-covalent bonds outside the recognition helix-base pair contacts do?

stabilizes interactions between DNA & transcription factor by binding to the sugar-phosphate backbone of the DNA helix

what is advantageous about the structure/orientation of the recognition α-helix (AKA second α-helix)?

it is a transcription factor within the helix-turn-helix motif that sticks out & down into the grooves of DNA where it has access to nitrogenous bases that it can read & bind to

which base pairs can the major groove of a DNA transcription factor differentiate between?

A=T & T=A

G≡C & C≡G

which base pairs can the minor groove of a DNA transcription factor differentiate between?

A=T & T=A

BUT NOT G≡C & C≡G

why do bacterial transcription factors mostly bind DNA with dimers?

- since both subunits of the dimer contain a helix-turn-helix motif, each subunit has a recognition helix

- increases specificity & stability of interaction

what are the 4 mechanisms of negative gene regulation?

- negative regulation

- repressors

- operators

- effectors

define negative regulation:

repressor protein blocks transcription

define repressors:

proteins that block RNA polymerase binding or its movement along DNA

define operators:

binding sites on the DNA that bind repressors (generally located near a promoter)

define effectors:

a small molecule binds to repressor causing a conformational change to regulate DNA binding

what are the 4 mechanisms of positive gene regulation?

- positive regulation

- activators

- activator-binding sites

- effector

define positive regulation:

regulation by means of an activator protein that induces transcription

define activators:

proteins that enhance the RNA polymerase-promoter interaction

define activator-binding sites:

often adjacent to promoters that are bound weakly or not at all by RNA polymerase alone