MOLEC 2: Exam 1

The Central Dogma (old view)

info cant be transferred back from protein to either protein or nucleic acid

The Central Dogma (modern view)

general flow of sequence information from genes to proteins

retroviruses

Use RNA-dependent DNAPol to integrate RNA genome via reverse transcription

RNA genome replication, RNA amplification and RNA dependent RNAPol are used by what?

coronaviruses, retroviruses, many euk.

who coined the idea of “one gene, one enzyme”

Beadle

Avery, MacLeod, and McCarty discovered what? what microbe were they studying?

DNA is the repository of genetic information. Streptococcus pneumoniae

genes that can encode different proteins are called

splicing genes

ORF stands for

open reading frame

CDSs stands for

Coding DNA sequences

what are genes

DNA segments that contribute to phenotype/function

describe what an open reading frame (ORF) is? describe the structure.

• section of RNA that can encode a protein

• contains a continuous series of non-overlapping codons

• Start codon → aa codon →stop codon

Prokaryotic polycistronic mRNA can only be found in prok

false, rare in euk

polycistronic mRNA

• one mRNA can be translated into multiple different proteins

• contains multiple ORFs

• apart of an operon

• genes encode enzymes involved in consecutive steps of the same metabolic pathway.

what protein the start codon encode

Met

Monocistronic mRNA

• contains one ORF

• encodes ONE protein

• 1 mRNA →1 protein

• contains 1 promoter, which is individually regulated

monocistronic mRNA structure

5’ cap—[ 5’-UTR— ORF1— 3’-UTR—] —3’-polyA

define operon

DNA encoding polycistronic mRNA

T/F: Operons are under control of multiple promoters

F: under control of a single promoter for transcription

Prok gene expression and regulation

• quickly adapts to enviornment

• short mRNA half life

• simple regulation (operon)

• genes are organized in operons

Euk gene expression and regulation

• intricate regulation → allows cell differentiation and development

• Operons are rare (1 gene = 1 promotor)

• long mRNA half life

• spatiotemporal regulation

what two key aspects of euk gene regulation allow for cell differentiation and specialization?

1) adaptations in enviornmental changes (stimuli response)

2) spatiotemporal regulation

define spatiotemporal regulation

where specific genes are activated at precise times and locations

what direction does the coding strand follow

5’ →3’

what direction does the template strand follow

5’ →3’

T/F: the coding strand follows the same sequence as the DNA temple

T: with the exception that T is replaced with U

what 4 energy molecules required for RNA synthesis by RNApol?

• ATP

• CTP

• GTP

• UTP

what direction does the RNA chain grow in?

5’→3’

what determines the RNA sequence?

DNA template sequence

T/F: RNA sequence is complementary to template strand

T: complementary base pairing

what number indicates the transcription site/start point?

+1

upstream digits reflect what type of integer?

negative integers

downstream digits reflect what type of integer?

positive integers

when does transcription start?

When RNAPol binds to the promoter regions of a gene

define transcription unit

strech of DNA that is transcribed by RNAPol from the start site to termination site (may include more than one gene)

T/F: a transcription unit can contain one or multiple genes

T

Transcription synthesizes what?

complementary strands of mRNA, composed of ribonucleotides (NTPs)

which of the following does NOT require a primer?

• RNA polymerase

• DNA polymerase

RNA polymerase

which of the following is more error prone?

• RNA polymerase

• DNA polymerase

RNA polymerase

Transcription can be considered to be replication to a portion of a genome how many times?

multiple times

replication can be considered replication of an entire genome how many times?

once

whats the error rate of DNA polymerase

10^-9 - 10^-10

whats the error rate of RNA polymerase

10^-4 - 10^-6

differences between DNA and RNA pol

• RNA pol: no mismatch repair mechs after RNA/protein syn

• DNA pol: contains post synthesis mismatch repair mechanisms

  • BOTH: have proofreading capabilities

T/F: Proteins and RNA can be produced with lower accuracy with long-term consequences

F: they can be produced with lower accuracy without long-term consequences

T/F: DNA replication does NOT require high accuracy to maintain inheritance of function

F: it DOES require high accuracy, but occasional errors → evolution

describe where Transcription and translation happen in prok and euk

• euk

  • Transcription→nucleus

  • Translation →ribosomes in cytoplasm

• prok:

  • Transcription →cytoplasm

  • Translation →cytoplasm on ribosomes

describe the flow of transcription for euk

Nucleus: DNA →transcription →pre-mRNA →RNA processing →mature mRNA

describe the flow of transcription for prok

Cytoplasm: DNA →transcription →mRNA

which of the following is a type of coding RNA

• snRNA

• snoRNA

• mRNA

• tRNA

• regulatory RNAs

• rRNA

mRNA (ONLY CODING RNA)

define snoRNA

small nucleolar RNAs used to process and chemically modify rRNAs; important in making functional ribosomes

define snRNA

small non-coding RNA found in the nucelus helps with RNA splicing; combine with proteins to form snRNPs (small nuclear ribonucleoproteins)

Define spliceosome

RNA-protein complex that performs RNA splicing; removes introns from pre-mRNA and joins exons together to form mature mRNA

snRNPs is made up of what?

snRNA + proteins

describe the flow of mRNA synthesis

pre-mRNA →spliceosome removes introns → exons joined →mature mRNA

define primary transcript

original unmodified RNA product corresponding to a transcription unit

the template strand is also called what?

antisense sense

the coding strand is also called what?

sense strand

what 3 steps are involved in transcription

1. Initiation

2. elongation

3. termination

Prok. RNA polymerase is a core enzyme, list and describe the subunits

• α₂ : (two alpha subunits) helps with enzyme assembly and interactions with regulatory proteins

• β: (beta) helps form active site

• β’: (beta prime) with with β to form active site “princers/crab claw”

• ω: (omega) helps with RNA pol assembly (not essential in bacteria)

T/F: can RNA pol initiate transcription as a core enzyme

F: cant initiate transcription as a core enzyme, but as a holoenzyme it can

Can Prok. RNA pol start transcription with an intact dsDNA template?

no, only ssDNA and nicked DNA templates

describe the function of the σ factor

• increases specificity with the promoter

• promoter recognition

• promotes isomerization (DNA melting)

what is the prok core promoter made up of?

the -10 and -35 motifs, optional UP-element

what σ factor binds to the -35 and -10 regions of the promoter

• σ₄: -35

• σ₂: -10 (pribnow box)

T/F: promoters are not well conserved in prok.

T: some promoters are stronger than others, consensus seq are typically strong promoters

define regulon

genes in bacteria that coordinate control of a single regulatory mechanism (ex: σ₇₀)

what is σ₇₀ called? what does it do?

• housekeeping sigma factor

• promotes expression of most genes, and essential genes for exponential growth

why is transcription specificity different in prok?

different σ factors recognize different regions of consensus seq for the expression of regulons

what subunit of the σ provides DNA melting energy to prok. RNA pol? and why?

• σ₂

• because its A/T rich

what is more stable: Helix-turn-Helix or Helix-loop-Helix

helix-turn-helix

define the isomerization step

where the RNA pol holoenzyme melts the DNA to create an open complex for transcription to begin

what one of three steps of translation does the isomerization step take place?

step 1- initation

T/F: discriminators can determine promoter strength in prok translation

T: extended -10 element

describe abortive initiation

where RNA pol repeatedly makes short RNAs before successfully leaving the promoter and entering elongation. needs ~10 nt to escape promoter.

what is the rate limiting step for transcription?

isomerization

Promoter clearance describes what event?

sigma factor dissociation from RNA Pol core

T/F: abortive initation happens ONLY in prok

F: found in all RNA Pol

T/F: viruses have RNA pol

T: Yes, monomeric (bacteriophage T7)

What drug is used to inhibit transcription in Prok?

Rifampicin

• bactericidal

• blocks RNA exit tunnel = RNApol remains stuck at promoter

describe prok transcription initiation

Promoter recognition →Closed complex forms (RNA pol bound to promoter) →Isomerization →open complex forms →abortive initiation →promoter clearence → elongation begins

T/F: σ₅₄ RNA pol req and activator protein

T: σ₅₄ nitrogen metabolism regulon (protein = IHF)

define activator proteins

ATP dependent thetranscription factors that increase transcription efficiency

EBPs stand for? what are the also known as?

Enhancer binding proteins, aka activators

what ions bind to the “active center cleft” of the active site?

(2) Mg²⁺

T/F: the two-metal ion catalytic mechanism is seen ONLY in Prok RNApol

F: found in all types of polymerases

T/F: elongation Is reversible

T: reversible via translocation →Brownian ratchet mechanism

how many Mg²⁺ ions are bound to one incoming nucleotides

1 Mg²⁺ molecule, the other comes from the RNA strand

list the 3 channels of RNA pol elongation complex

• duplex-binding channel

• RNA exit channel

• NTP channel

describe the function of the Duplex-binding channel

• holds incoming dsDNA and helps position it for strand separation

• creates a 90 degree turn

• helps create a RNA-DNA hybrid

• formed by β' subunit

• DNA strand separation begind around +2

describe the function of the RNA exit channel

interacts with the ssRNA from the RNA-DNA hybrid to guide it out of the polymerase

what eliminates supercoils?

Topoisomerases

define processivity

ability to perform consecutive reactions without dissociation

describe the function NTP channels

where NTPs are bound to one Mg²⁺

T7 RNA Pol has a simple promoter what is it?

specificity for beta-sheet interactions in the -7 and -11 grooves

what can be used for in vitro transcription?

T7 RNA Pol

what can be used for in vivo protein expression?

pET plasmids

describe the concept of pET vector

Gene of interest →cloned into pET plasmid behind T7 promoter →T7 RNA Pol expressed →strong transcription → lots of proteins

why might a pET vector may be useful?

when proteins being studied are toxic to the bacteria

what are 2 proofreading mechanisms are seen in elongation?

1) pyrophosphorolytic editing

2) hydrolytic editing

describe pyrophosphorolytic editing

type of proofreading mechanisms during elongation, that removes single Incorporated nucleotide using PPi