Molecular genetics | Quizlet

Function of DNA polymerases

what is the difference in priming which is necessary for DNA replication in reciprocates and Eukaryotas?why?

Bacteria involves less proteins

Having more proteins allows more regulation which is more necessary for eukaryotes

what are functions of E.coli polymerases I,II,III

polymerases i,iii are found only in prokaryotes

polymerase I= deletes RNA primers and replaces them with DNA,synthesis DNA but the main DNA synthesis is done by DNA pol.iii

polymerase III=main replicatory enzyme,is multisubunit protein

what does topoisomerases do?How many types are there and what are differences(in eukaryotes)?

Toposiomerases cut DNA(mostly for relief of torsional tension or generationg negative supercoils):

IA = makes a nick in one of the strands in a way that uncut strand passes through it once

IB = makes a cut(nick) in one strand but also rotates a cut strand around uncut one

IIA

IIB

what is nick?

a gap between two adjacent nucleotides in which there is no phosphodiester bond

What is the linking number?

number of times one strand(cut by topoisomerase I) winds around the other. For closed circles only.

How does initiation of replication happen in yeast?

Origin recognition sequence.made of ACS(autonomous consensus sequence) and subdomain B1

ORS=ACS+B1

in yeast.It is imporntant to build up a protein complex to recognise oringin of replication and proteins to melt DNA to start replication(p365 down)

why are regions which are rich in A-T bonds are called melting regions?

because number of H bonds between them is always 2 which makes it easy to break and initiate for example replication

how many H bonds are formed between A and T,C and G?

2between A and T

3 between C and G

how does initiation of replication happens in prokaryote?

Origin of replication is oriC

AT-rich DNA unwinding element(DUE) and 5-12 binding sites with diff affinity for proteins DnaA which negatively supercoil DNA.

AT-rich DUE melts because of torsional stress which initiates replication fork and attachment of prepriming complex(2 of them=1 per side of replication fork)(p365)

what is origin of replication?

origin of replication=it is a place where origin recognition complex is build up,and where it will start replication

Ch 9.2 What is transposition?What is the difference between conservative and replicative transposition?

=the process by which a segment of DNA can move from one position to another in a genome. These movable segments are called transposable elements or transposons.

During conservative transposition results in the transposon simply changing its position in the genome without increasing its copy number .

Replicative transposition, on the other hand, results in an increase in copy number, since the original element remains in place while a copy is inserted at the new position.

what are transposable elements or transposons?

There is a process such as transposition=the process by which a segment of DNA can move from one position to another in a genome. These movable segments are called transposable elements or transposons.

Does transposition involve recombination?

yes

what are homologous recombinations?

What is SSB and what does it do(genome replication)?

What is a frameshift mutation?

mutation that shifts the "reading" frame of the genetic message by inserting or deleting a nucleotide(not a multiple of 3,so codons downstream the genes are different which leads for them coding for different amino acids which in turn may make protein nonfunctional)

Describe retrotransposition

transposition(single strand recombination) which involves RNA intermediate

starts with synthesis of an RNA copy form DNA of the retrotransposon by the normal process of transcription (Figure 9.14). The transcript is then copied into double-stranded DNA, which initially exists as an independent molecule outside of the genome. Finally, the DNA copy of the transposon integrates into the genome, possibly back into the same chromosome occupied by the original unit or possibly into a different chromosome. So we end up with multiple copies in the genome.

what distinctions are there between different replicative transposons(=Homologous recombinations)?

process itself involves RNA intermediate or it does not.

The process which involves RNA called retrotransposition.It involves synthesis of an RNA copy form DNA of the retrotransposon by the normal process of transcription (Figure 9.14). The transcript is then copied into double-stranded DNA, which initially exists as an independent molecule outside of the genome. Finally, the

DNA copy of the transposon integrates into the genome, possibly back into the same chromosome occupied by the original unit or possibly into a different chromosome. So we end up with multiple copies in the genome.

what are endogenous retroviruses(ERVs)?

Evolutionary remnants of retroviruses that are silent parts of our genome.they are more decayed viral retroelements than transposones and they make about 9% of human genome.

Have long terminal repeats(LTR)

However some of them are active retrotransposomes if different individuals are compeared and those retroviruses can lead to disorders if esential genes are diturbed(like in frameshift mutation)

what is difference between two types RNA transposomes(retroelements)?

Some of them have LTRs(=long terminal repeats) and some of them don't

what are the names of RNA transposones which don't have LTRs(long terminal repeats)?

retroposones:

LINEs(long interspersed nuclear elements)

SINEs (short interspersed nuclear elements).

out of three LINEs(long interspersed nuclear elements) families which one still can transpose(other two are inactive relics)

LINE1

what is LINE1?

one of three LINEs(long interspersed nuclear elements) families which one still can transpose(other two are inactive relics)

why are LTRs(long terminal repeats) are important for RNA transposones?

Long terminal repeats do play a central role in the process by which the RNA copy of an LTR element is reverse-transcribed into double-stranded DNA (Section 17.3), are also possessed by viral retroelements (see Figure 9.7).

SINEs (short interspersed nuclear elements) do not have their reverse transcriptase and not having LTRS(long terminal repeats) how do they deal with it?

they borrow reverse transcriptases which have been synthesised by LINEs(long interspersed nuclear elements)

which element is shared by both LINEs and SINEs?

poly(A) sequence(and of course poly(T) sequence on the opposite strand

What is the Alu?

The commonest SINE in primate genomes

what is SINEs

short interspersed nuclear elements=RNA transposon without LTR(long terminal trpeats).

borrows reverse transcriptases which have been synthesised by LINEs(long interspersed nuclear elements which also is RNA transposon without LTR(long terminal trpeats).)

Are RNA transposones more common in eukaryotic or prokaryotic organisms?

in eukaryotic organisms

what is transposase ?

enzyme that catalyzes its transposition for DNA transposones(no reverse transcriptase involved)

name 4 types of DNA transposons in prokaryotes

(p213-214)

Insertion sequence

Composite transposon

Tn3-type transposon

Transposable phage

what is IS(insertion sequence) in DNA transposons?(p214)

Bacterial insertion sequences. Insertion sequences, or insertion-sequence (IS) elements, are now known to be segments of bacterial DNA that can move from one position on a chromosome to a different position on the same chromosome or on a different chromosome.

(There is a pair of inverted repeats at either end of each IS element, up to about 50 bp in length depending on the type of IS, and insertion of the element into the target DNA creates a pair of short (usually 4-15 bp) direct repeats in the host genome. IS elements can transpose either replicatively or conservatively.)

what are composite transosons(DNA transposons)?(p214)

composite transposons are made up of a pair of IS(insertion sequence) elements flanking a segment of DNA, usually containing one or more genes, often ones coding for antibiotic resistance

what kind of transpsons are dangerous in terms of spreading antibiotic resistance genes?(p214)

composite transposons(which are part of DNA transposons)

what is a distinctive feature of Tn3-type or unit transposons (p214)

Tn3-type or unit transposons have their own transposase gene and so do not require flanking IS elements in order to transpose (Figure 9.19C). Tn3 elements transpose replicatively.

what are transposable phages?

are bacterial viruses that transpose replicatively as

part of their normal infection cycle p2013-2014

are DNA transosones more common in prokaryote or eukaryote?

much more common in prokaryote

TIR

Two transposases recognize and bind to TIR sequences, join together and promote DNA double-strand cleavage. The DNA-transposase complex then inserts its DNA cargo at specific DNA motifs elsewhere in the genome, creating short TSDs upon integration

why are wild flies and lab flies can not produce fertile offspring?

The P element, a DNA transposon in Drosophila

melanogaster, was similarly discovered from studies of an unusual genetic

event that, as it turns out, arises from transposition. This event is called hybrid

dysgenesis and occurs when females from laboratory strains of D. melanogaster

are crossed with males from wild populations. The offspring resulting from

such crosses are sterile and have chromosomal abnormalities along with a

variety of other genetic malfunctions. The explanation is that the genomes of

wild fruit flies contain inactive versions of P elements, which are typical DNA

transposons comprising a transposase gene flanked by inverted terminal repeats,

but that laboratory strains lack these P elements. After crossing, the elements

inherited from the wild flies become active in the fertilized eggs, transposing

into various new positions and causing the gene disruptions that characterize

hybrid dysgenesis (Figure 9.22). Exactly why this activation occurs is not known,

but a more interesting question is why the genomes of wild populations of

D. melanogaster contain P elements whereas laboratory strains do not. Most of

the laboratory strains are descended from flies collected by Thomas Hunt Morgan

some 90 years ago and used by Morgan and his colleagues in the first gene

mapping experiments (Section 3.3). It appears that wild populations at that time

lacked P elements, which have somehow proliferated in wild genomes during the

last 90 years.

What is site-specific recombination?

A recombination which is initiated between two DNA molecules that have only very short sequences in common

what are 3 elements needed for transcription to happen?

1. Enzyme (DNA-dependent RNA polymerase)

2. Template (DNA)

3. Substrate (nucleosides triphosphate, or NTPs)

what is Transcription unit?

section of DNA that can be transcribed(may contain diff. n of genes)

Name general structure of a transcription unit from 5'to 3' and explain their role

Promoter=RNA polymerase(or transcription factors in eukar.) recognises it

Transcription start site(TSS)=region of DNA just after Promotor.First section of DNA to be transcribed.

Gene body

Terminator=section of DNA that ''tells'' RNA polymerase to stop to transcribe

Transcription end site(TES)=point,after terminator after which no DNA is transcribed

Name general structure of a transcription unit from 5'to 3'

Promoter

Transcription start site(TSS)

Gene body

Terminator

Transcription end site(TES)

what is the role of Promoter in transcription?

The promoter is essential for transcription initiation .RNA polymerase(or transcription factors in eukar.) recognises it and after that starts its activity

what is Transcription start site(TSS)?

Transcription start site(TSS)=region of DNA just after Promotor.First section of DNA to be transcribed.

what is the role of Terminator in transcription?

section of DNA that ''tells'' RNA polymerase to stop to transcribe

what is Transcription end site(TES)?

point,after terminator after which no DNA is transcribed

what does a promotor contain?

A typical promoter contains one or more specific short

DNA sequence elements (motifs) that are recognized

either by the RNA polymerase or by other accessory DNA

binding proteins

what are 4 subunits that constitute the core enzyme in Polimerase in bacterea?

1. (2X) α subunits (essential for enzyme's assembly)

2. β and β' subunits (constituting the active site responsible for RNA synthesis)

3. ω subunit (assembly and stability of the enzyme)

• A 5th subunit, called σ, is the transcription initiation factor

• The core enzyme associates with the σ factor to form the

RNA polymerase holoenzyme

• It provides sequence specificity to the RNA polymerase,

thus allowing specific binding to the promoter

• Bacteria can change the specificity of the RNA

polymerase by changing the σ factor

• The core enzyme has a general (non-specific) affinity for

DNA

what are two typical bacterial promoter motifs?

1. -10 box (σ70 consensus sequence: TATAAT

2. -35 box (σ70 consensus sequence: TTGACA)

why can the affinity of the RNA polymerase holoenzyme for the promoter vary?

Variations in the -35 and -10 box sequences affect the affinity of the RNA polymerase holoenzyme for the promoter, thus the

efficiency of transcription initiation

what is the most studied examples of regulation of transcription in

bacteria?

lactose operon

How is transcription in bacteria regulated(in general terms)?

Synthesis by RNA polymerase is regulated by several

DNA binding proteins

• These proteins can act both as activators (positive

regulation) or repressors (negative regulators)

• Transcriptional regulation allows bacteria to tightly control

gene transcription, in order to transcribe specific genes

only when it is strictly needed

• One of the most studied examples of this regulation in

bacteria is the lactose operon

Synthesis by RNA polymerase is regulated by several

DNA binding proteins.Name two roles that those proteins have

These proteins can act both as activators (positive

regulation) or repressors (negative regulators)

describe the work principle behid lac operon

• genes for the metabolism of lactose are only transcribed when lactose is present

• The lac operon is composed of a promoter, controlling the

expression of genes needed for lactose metabolism, and an operator

• The operator is a sequence recognized by a repressor

protein (lacI)

• The repressor is constantly transcribed at basal levels

• In the absence of lactose, the repressor binds to the operator, competing with the RNA polymerase, hence inhibiting transcription

• In the presence of lactose, allolactose (an isomer of lactose) binds to the repressor, inducing a conformational change that impairs the ability of the repressor to bind the operator

• The RNA polymerase is then free to transcribe the genes needed for the metabolism of lactose

What is an operator?

a specific region of the DNA at the initial end of a gene or operon, where a repressor protein binds and blocks mRNA synthesis

what is operon

A set of genes transcribed under the control of an operator gene. More specifically, an operon is a segment of DNA containing adjacent genes including structural genes, an operator gene, and a regulator. An operon is thus a functional unit of transcription and genetic regulation.

why are there so many mechanisms to regulate gene expression?

to ensure that cell will transcribe genes when they are needed and in amount needed to be as efficient as possible by using promotors and repressors.(for ex. lac operon)

what is the role of repressor protein during transcription?

repressor protein binds to a specific region of the DNA= operon, a and blocks mRNA synthesis

what is the difference between operator and operon

Operator is a segment of DNA to which a transcription factor binds to regulate gene expression by repressing it. The protein that does this is called a repressor.

Operon: A set of genes transcribed under the control of an operator gene. More specifically, an operon is a segment of DNA containing adjacent genes including structural genes, an operator gene, and a regulator. An operon is thus a functional unit of transcription and genetic regulation.

Therefore operator is part of operon

what is transcription termination?

Transcription termination is the process by which the RNA Polymerase dissociates from the template DNA

name Two main types of transcription terminators in bacteria:

1. Intrinsic (or Rho-independent)

2. Rho-dependent

describe Intrinsic (Rho-independent) transcription termination( in bacterial transcription)

• A stem-loop(hairpin) structure is formed by freshly transcribed RNA folding into itself, causing RNA Polymerase to pause

• A stretch of A residues is transcribed right before RNA Polymerase pausing

• This stretch of A-U pairs weakens the interaction between the nascent RNA and the template DNA, leading to detachment of the RNA Polymerase

describe Rho-dependent terminators(in bacterial transcription)

• Rho helicase (ATP-dependent melting of nucleic acids)

• Rho is needed to resolve the base-pairing between the

template DNA and the nascent RNA, with consequent

detachment of the RNA polymerase

name 3 dif RNA Polymerase enzymes and what specialisations/roles they have

1. RNA Polymerase I (rRNA genes)

2. RNA Polymerase II (mRNAs, certain snRNAs and

snoRNAs, miRNAs, etc.)

3. RNA Polymerase III (tRNAs, certain snRNAs and

snoRNAs, 5S rRNA, etc.)

• The promoters recognized by each RNA Polymerase

enzyme have distinct structural features

which RNA polymerase specialises in mRNA synthesos

RNA Polymerase II

are promoters for different RNA polymerases same?

No, The promoters recognized by each RNA Polymerase

enzyme have distinct structural features

what is the most imporntant core promoter for RNA Polymerase II?

TATA box (consensus: TATAWAAR)

what is the difference between RNA polymerase II and bacterial RNA polymerases?

Unlike bacterial the RNA Polymerase, RNA Polymerase II does not bind directly to the core promoter

• A set of accessory transcription factors (TFs, DNA binding proteins) mediates RNA Polymerase binding

• The TFIID complex, composed of a TATA-binding protein (TBP) and several TBP-associated factors (TAFs), mediates the initial recognition

What are transcription factors?

regulatory proteins that bind to specific DNA sequences(in eukaryotes RNA polymerases do not bind to DNA directly but do so via transcription factors as mediators)

describe preinitiation complex (PIC) for eukaryotes(use RNA polym II)

• RNA Polymerase II, together with other 5 TFs, is positioned on this platform, forming the preinitiation complex (PIC)

• The C-terminal domain (CTD) tail of the RNA Polymerase presents 52 repeats of the sequence Tyr-SerPro-Thr-Ser-Pro-Ser

• The TFIIH factor of the PIC has kinase activity and catalyzes the phosphorylation of Ser 5 in the CTD of

• Phosphorylation causes the release of the RNA Polymerase from the elongation complex and the transcription initiation

What is the preinitiation complex (PIC) (transcription)?

• RNA Polymerase II, together with other 5 TFs, is positioned on this platform, forming the preinitiation complex (PIC)

• Phosphorylation of amino acids on C terminal causes the release of the RNA Polymerase from the elongation complex and the transcription initiation

from which terminus N or C aminoacids are red?

N -teminus is the beginning so from N terminus to C terminus

Besides core TFs(translation factors) involved in transcription initiation,several other TFs can tightly regulate transcription by minding to genes.Name two kinds of TFs binding

1. Proximal (usually within 2Kb of the TSS, promoter)

The TF only regulates the nearby gene

2. Distal (up to several Kb or Mb, enhancer)

The TF can regulate multiple genes

what is mediator and its role in transcription?

An adaptor protein, called Mediator, transduces (or mediates) the signal from the TF to the PIC complex(better see pic in the end of transcription slides)

why are TFs(transcription factors) necessary for regulation of transcription?

• Transcription initiation in the absence of accessory TFs is very

weak (low basal levels of transcription)

• Most known TFs act as activators, increasing the efficiency of transcription initiation

• A few known TFs act as repressors, inhibiting transcription initiation

most transcription factors act as ... and minority as ...

activators and minority as repressoes

what are Three sequence determinants are essential for splicing?

1. Donor site First 2 bases of the intron, usually GU

2. Branch point An A residue, close to intron's 3' end

3. Acceptor site Last 2 bases of the intron, usually AG

of thos two donor and acceptor are most imporntant

Describe RNA splicing

• First cut occurs at the level of the donor site

• This is mediated by the nucleophilic attack by the C2

hydroxyl (OH) of the A residue at the branch site, to the

phosphate of the RNA backbone, right upstream of the

donor site (transesterification)

• This create a structure in which the intron loops back on

itself, called lariat intron

A second transesterification occurs between the 3'-OH

group of the upstream exon and the phosphate group of

the RNA backbone, right downstream of the acceptor

site

• The lariat intron is linearized and degraded

give general characteristics of splicing

• Splicing involves very simple chemical reactions

• Biggest challenge for the cell is to deal with the distance

between splicing sites (up to hundreds of Kb in some

cases)

• A ribonucleoprotein (RNP) complex, called

spliceosome, is involved in bringing the splicing sites in

close proximity, hence aiding the splicing process

what is the role of splisosome?

to bring donor and acceptor together to do transesterification

what is role of LTRs(long term repearts) in viruses?

they act as promotors in viruses

do DNA transposones transpose replicatively or conservatively?

they transpose only replicatively

Transposition and site-specific recombination are two categories of DNA rearrangements that are mediated by specialised enzymes generically referred to as transposases and site-specific

recombinases.

what is the difference between Transposition and site-specific recombination?

Transposition is the process by which genetic elements move between different genome locations, whereas site-specific recombination is a reaction in which DNA strands are broken and rejoined at determined positions of two target DNA sequences.

Describe lysogenic and lythic infection cycle

After bacterial phage is injected in DNA:

Lytic infection cycle:

DNA directs synthesis of new phages

New phages are released,destroying the cell

Lysogenic infection cycle:

1.Bacteriophage DNA is integrated into the bacterial chromosome

2.Many cell divisions

3.DNA directs synthesis of new phages

4.New phages are released,destroying the cell

between what kind of DNA molecules does Homologous recombination takes place?

between diff. DNA molecules with extensive sequence homology

or within single molecule

what are requirments for SSR(site specific recombination)?

two very different DNA molecules with a short region of homology

what would be consequences if recombination would not exist?

Without recombination genomes would be relatively static with few changes

Gradual accumulation of mutations 🡪 small-scale alterations in genome nt sequence

But no major restructuring

Evolutionary potential of genome severely restricted

Homologous recombination: Holliday, Meselson-Radding,Double-strand break model .Which one of those models is currently accepted in science?

Double-strand break model for homologous recombination is currently accepted

What are Nicks?which enzyme(s) make them?

deskribe step by step ,Double-strand break model for homologous recombination

1.one of two double strands is cut by exonuclease (sticky ends are formed)

2.the 3' side of cut DNA invades uncut molecule,forming H-bonds between itself and invaded DNA nucleotides(strand invasion)(

3.Invading strand + partner are extended by DNA polymerase (5'🡪3').(uncut DNA forms H bonds with another DNA strand which was cut)

4.Ligase joins the gap,so we get Heteroduplex with two Holliday junctions

5.Cleawage of Holliday juctions(resolution)

6.Mismatch repair

what is now though to be the most important role of Homologous recombination?

it is though to an important mechanism in DNA repair

What is gene conversion?

Non-reciprocal transfer of information between homologous sequences

how is gene conversion is beneficial to pathogenic microorganisms?

In pathogenic bacteria:

- generation of antigenic variation, allowing to avoid host immune system

Homologous recombination in E. coli: the RecBCD pathway

RecBCD pathway(p416-417)

RecBCD attaches to double-strand break

RecB (helicase and 3ʹ→ 5ʹ exonuclease):degrades strand it is traveling along (the one with free 3ʹ-end) until chi site

RecD helicase detaches.This stops RecB exonuclease activity

RecB makes single cut in other strand near chi in a way that DNA molecule with 3ʹ-overhang

Heteroduplex formation Mediated by RecA

RuvAB: a molecular motor, rotating helices so that branch point moves"making heteroduplex"(you know like X letter)

RuvC: resolvase, cleaves and resolves Holliday structure

describe role of RecBCD in homologous recombination in E.coli(p416)

Translocation of the RecBCD complex along the DNA is accompanied by 3ʹ→ 5ʹ degradation of the upper strand, due to the exonuclease activity of RecB.

When a chi site is encountered, the exonuclease activity is suppressed and the RecB endonuclease cleaves the lower strand, giving the 3ʹ-overhang. If RecBCD translocates some distance along the DNA before reaching a chi site, then it is likely that the 5ʹ- overhang shown in the second panel will occasionally be cleaved

by the endonuclease, so that this overhang is never more than a few tens of base pairs in length.

RecA proteins bind to overhang and use it to do strand invasion,forming a D-loop

After that Branch migration is catalyzed by the RuvA and RuvB proteins, both of which attach to the branch point of the heteroduplex formed by invasion of the 3ʹ-overhang into the partner molecule.

After branch migration, two RuvC proteins binds to the junction, the orientation of their attachment determining the direction of the cuts that resolve the structure.

what is role of Ruv proteins in homologous recombination in E.coli?(p417)

After RecBCD pathway and formation of D-loop by RecA, migration is catalysed by the RuvA and RuvB proteins, both of which attach to the branch point of the heteroduplex formed by invasion of the 3ʹ-overhang into the partner molecule.

After branch migration, two RuvC proteins binds to the junction, the orientation of their attachment determining the direction of the cuts that resolve the structure.

what is the role of RecA protein in homologous recombination in E.coli?(p416)

After RecBCD pathway,RecA proteins bind to overhang and use it to do strand invasion,forming a D-loop