Dudi Engelberg
This presentation is the property of its rightful owner.
Sponsored Links
1 / 145

Dudi Engelberg Room 1-517 Tel: 658 4718 e-mail: [email protected] PowerPoint PPT Presentation


  • 43 Views
  • Uploaded on
  • Presentation posted in: General

Dudi Engelberg Room 1-517 Tel: 658 4718 e-mail: [email protected] The central dogma of molecular biology. DNA. Transcription. RNA. Translation. Protein. Could proteins multiply ?. What do we have RNA for?. Same DNA content in all cells of the mulicellular organism?

Download Presentation

Dudi Engelberg Room 1-517 Tel: 658 4718 e-mail: [email protected]

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Dudi Engelberg

Room 1-517

Tel: 658 4718

e-mail: [email protected]


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

The central dogma

of molecular biology


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

DNA

Transcription

RNA

Translation

Protein


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Could proteins multiply ?


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

What do we have RNA for?


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Same DNA content in all cells of the

mulicellular organism?

What is the function of DNA?

Can cells function without DNA?


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Are these all nucleotides that appear in DNA and RNA?


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

What are the cellular functions of nucleotides?


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Some cellular functions of nucleotides

Building blocks of nucleic acids.

2. Energy carrier (ATP, GTP).

3. Building parts of enzymes co-factors (e.g., NAD, FAD,

CoenzymeA, S-adenosylmethionine).

4. Regulators in signal transduction processes.

5. Second messengers in signal transduction (cAMP, cGMP).

6. Phosphate donors in phosphorylation reactions. Involved in many

more pottranslational modifications.

7. Serve as structural molecules (rRNA).

8. Activators of carbohydrates for synthesis (glycogen for example).


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Some cellular functions of deoxynucleotides

Building blocks of nucleic acids (DNA).


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Some cellular functions of deoxynucleotides

Building blocks of nucleic acids (DNA).

2. Energy carrier (ATP, GTP).

3. Building parts of enzymes co-factors (e.g., NAD, FAD,

CoenzymeA, S-adenosylmethionine).

4. Regulators in signal transduction processes (GTP).

5. Second messengers in signal transduction (cAMP, cGMP).

6. Phosphate donors in phosphorylation reactions.

7. Serve as structural molecules (rRNA).

8. Activators of carbohydrates for synthesis (glycogen for example).


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Some deviations from the averaged Watson & Crick model

The pitch angle between base pairs could be 28o - 42o.

Bases could propel (deviate from planarity).

Damages: kinks and covalent bonding inside the helix (usually

Between bases).

Presence of unusual bases (in tRNA for example) allows unusual

base pairing and novel structural motifs.

Presence of specific sequences (stretch of purines,

palindromes, sequence repeats).


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

The driving force towards

synthesis is the breakdown of

PPi.

Phosphodiester bond


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Mechanism of the basic synthesis reaction of nucleic acids

Addition of nucleotide involves an attack by the 3’-hydroxyl group at the end of the growing RNA molecule on the a phosphate of the oncoming NTP.

Two Mg2+ ions coordinated to the phosphate groups of the NTP and to three Asp residues of the  subunit of E. coli RNA polymerase

(conserved in most RNA polymerasess in nature).

One Mg2+ ion facilitate the attack by the 3’-hydroxyl group on the a phosphate and the other ion facilitates the displacement of pyrophosphate.

The Mg2+ ions stabilize in fact the transition (intermediate) state.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Polymerization of nucleotides - DNA and RNA biosynthesis

1. The reaction is directional; proceeds from 5’end to 3’end.

As a result the product is asymetric (5’end different than

3’end.

2. The nucleotides (of the same strand) are always linked

in a phospho-di-ester bond (a covalent bond).

3. Energy is wasted in addition of each monomer. The

driving force towards synthesis is degradation of

pyrophosphate.

4. The precursors are always nucleotides tri-phosphates

(NTPs or dNTPs).

6. The reaction is directed by a pre exist plan (a template).

(No polymerase is capable of adding nucleotides randomly).

May be there are some - quite important


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Basic characteristics of DNA Pol

Is not capable of de novo synthesis.

Requires:

A. A template (as any other polymerase).

B. A primer (RNA oligo, nicked DNA, protein?)

Possesses two catalytic activities:

A. A 5’ to 3’ polymerase activity.

B. A 3’ to 5’ exonuclease actiivty.

3. Substrates are only dNTPs.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

How DNA Pol is regulated?

Does it possess regulatory

sites?


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

DNA replication is semi-conservative

DNA replication is bi-directional


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Schematic structure of E. coli replication origin (OriC)

245 bp.

3 repeats of 13 bp sequences + 4 repeats of 9 bp sequence.

These elements are highly conserved in replicationorigins

of bacteria.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Initiation step: “opening” DNA “preparing the template before

any DNA synthesis occurs.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

First key step in replication: binding of DnaA protein molecules

to the four 9 bp repeats.

DnaA binding requires ATP and HU

Second step: binding of DnaB (hexamerix helicase). Two hexamers bind to unwind

DNA at two points creating two potential replicating forks.

Third step: binding of SSBs (essential for stabilizing single strand throughout the

replication process) and DNA gyrase (DNA topoII) - this step allows DnaB

helicase to unwind thousands of base-pairs.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

DnaA binds cooperatively to form a core

around which OriC DNA is wrapped.

At the presence of ATP DnaA melts the

DNA of the A-T rich 13 bp tandem repeats.

DnaA molecules recruit two DnaB-DnaC

complexes, one for each replication forks.

(6 DnaC monomers bind the DnaB hexamer.)

Gyrase must be present to relieve topological

Stress - otherwise helicase cannot further

catalyze unwinding.

Altogether a pre-priming complex is formed:

480 kD, 6 nm radius.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Initiation step has prepared the template.

Moving to elongation step:

Priming is required.

A mechanism for bi-directionality is required.

Leading strand synthesis begins with

The synthesis of a short primer (10-60 n)

catalyzed by primase (DnaG - special

RNA Pol).


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Both strands are sybthesized by DNA Pol3.

Lagging strand:

A new primer is synthesized near

the replication fork.

Synthesis continues until the

Fragment extends as far as the primer

of the previous fragment.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Specific structural capabilities of

DNA Pol 3.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

DnaB (helicase) + DnaG (primase) form a functional unit

within the replication fork, called primosome.

DNA pol3 - a dimer - one set of subunits synthesize the

leading strand and other set the lagging strand.

Once DNA is unwound by DnaB, DnaG associates occasionally

with DnaB and synthesizes a short RNA primer.

A new  sliding clamp is then positioned at the primer by the

clamp-loading complex of Pol 3.

When a synthesis of a fragment is completed, replication

halts and the core subunits of Pol 3 dissociate from their 

sliding clamp and from the new fragment.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

 subunits on DNA


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Exonuclease activity is located

ahead of pol activity


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Sequence of the RNA is identical to that of the coding strand

(with the replacements of Us for Ts).


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Products of the transcription reaction (primary transcript):

In prokaryotes: an unstable RNA- rapidly degraded (mRNA

or cleaved to give mature products (rRNA, tRNA).

In eukaryotes: modified at the ends (mRNA) and/or cleaved

to give mature products (all RNAs).


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

With the exception of the RNA genomes

of certain viruses, all RNA molecules in

nature (mRNA, tRNA, rRNA, miRNA,

snRNA) are derived from information

stored in DNA and obtained

via transcription.

Namely, just like DNA during replication,

RNA is synthesized on DNA

template (DNA-dependent RNA synthesis).


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Transcription=DNA-dependent RNA synthesis


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Polymerization of nucleotides - DNA and RNA biosynthesis

1. The reaction is directional; proceeds from 5’end to 3’end.

As a result the product is asymetric (5’end different than

3’end.

2. The nucleotides (of the same strand) are always linked

in a phospho-di-ester bond (a saturated covalent bond).

3. Energy is consumed during addition of each monomer. The

driving force towards synthesis is degradation of

pyrophosphate.

4. The precursors are always nucleotides tri-phosphates

(NTPs or dNTPs).

6. The reaction is directed by a pre exist plan (a template).

No plymerase is capable of adding nucleotides randomly.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

At its basic enzymatic level, transcription is a

reaction highly similar to replication


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Comparison of replication to transcription (some aspects)

ReplicationTranscription

Quantity: The whole genome Parts of the genome

Timing: One time per life cycle some parts - all life time

(time is determined by the some parts - some time

checkpoint system) some parts - never

Location: From origin to end Many starts and many stops

(starts and stops must be

most accurate)

DNA substrate: The two strands One strand (could be a

different for each particular

case

Nucleotide

substrates: dNTPs NTPs


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Comparison of replication to transcription (some aspects)

ReplicationTranscription

Proofreading: Always Never

Post-reaction

repair: Always Never

Fate of

product: Remains attached to Released from

template template

Processivity: High or low High (from start to

termination)

Ligating

fragments: Yes No - products are independent molecules


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Sequence of the RNA is identical to that of the coding strand

(with the replacements of Us for Ts).


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Products of the transcription reaction (primary transcript):

In prokaryotes: an unstable RNA- rapidly degraded (mRNA

or cleaved to give mature products (rRNA, tRNA).

In eukaryotes: modified at the ends (mRNA) and/or cleaved

to give mature products (all RNAs).


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

RNA Polymerase - general properties

  • Properties similar to DNA Polymerases:

    - Basic chemical mechanisms: addition of ribonucleotides to the 3’-OH of the

    chain. Consequently determination of a 5’ to 3’ directionality.

  • Requires a template.

    - Adding nucleotides on the basis of optimal hydrogen bonds with the template

    strand (A-U, C-G).

    2. Properties specific to RNA Pol

    - Using only one strand as a template (must make a choice).

    - Does not require a primer (pppN 5’ end).

    - Very complex regulation for “choosing” the starting points (which may be

    different in every cell, in every developmental stage and in ageing.

    - Does not have a 3’ 5’ exonuclease activity.

    - The rate of mistake in high (1/104-105).


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

During a successful round: RNA Pol associates with the starting point and dissociates at the termination point, defining a transcription unit. A transcription unit may include more than one gene

Nomenclature: Upstream. Downstream; numbers; left to right; no base is defined as base zero.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Rates (in E. coli):

Transcription: 40 nuc../sec.

Similar to rate of translation.

Replication: 1,000nuc./sec/strand

RNA pol creates the

‘transcription bubble’ when

It binds to a DNA. The bubble

moves with it.

Displacing of the product

(RNA),

reforming the dsDNA


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

About 17 bp are unwound at any given time.

Length of RNA:DNA hybrid within the bubble: up to 12 bp.

Length of RNA within the bubble: ~25 b.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Within the transcriptional bubble (in bacteria), RNA Pol :

Unwinds and rewinds DNA

Maintains the conditions of the template and coding strands.

Synthesizes RNA.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

The transcription reaction can be divided into the

Following stages:

Template recognition - binding of RNA pol to DNA

at a sequence known as promoter forming a “closed

complex”, unwind the DNA to form an “open complex”,

creating the ‘bubble’.

Initiation - synthesis of the first nucleotide bond. RNA pol

Does not move while it synthesizes the first ~9 bases.

Abortive events may occur, forcing initiation to start again.

Initiation phase ends when the enzyme succeeds in extending

the chain and clears the promoter.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Elongation - enzymes moves along the DNA, extending the

RNA, unwinding the DNA exposing new segments of the

template and displace the RNA-DNA hybrid to re-form

the original double stranded DNA. RNA emerges as a free

single strand.

Termination - recognition of the point at which no further

bases should be added to the chain. The enzyme and the

RNA should be released and the DNA re-forms the original

duplex state.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Initiation of transcription: a crucial

(some time the only) regulatory

step in gene expression.

Some key questions:

How starting point is recognized?

How initiation rate is determined?

The transcription bubble: transiently

and shortly separation of the DNA

to single strands.

The process of transcription: the usual

complementary base pairing process.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Stages in which the bubble is created

Template recognition. Closed complex.

Local unwinding: open complex

(template strand is available)

Initiation (up to 9 bases that could be

released; no move)

Promoter clearance

Elongation - Movement of the bubble.

(inchworm move or fluent?)

Termination:1. Cease addition of nucleotides.

2. Set complex apart.

Just like initiation, termination is

sequence-dependent. Defines the terminator.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Promoter: The sequence of DNA needed for RNA

polymerase to bind to the template and accomplish

the initiation reaction (synthesis of the first nucleotide

bonds).

Terminator:The sequence of DNA required for

disrupting the bubble and reforming the DNA duplex

(after the last base is added).


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

 an ’ subunits have

a channel for the DNA


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Yeast RNA Pol II is composed of 12 subunits (holoenzyme). Two subunits form a different sub-complex. Two subunits are not essential for viability.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Following DNA binding and melting, the

“clamp” swings back to force a turn. [note, colors of subunits are

the same as in the crystal structure]


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

“wall” protein is enforcing

a turn.

The length of RNA hybrid

is limited by the activity of

the “rudder” protein. The

RNA is forced to leave the DNA

When it hits the protein rudder.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

The bridge protein is found in different conformations

In different crystal structures.

Probably, breaking and re-making of contacts

is mediated by conformational changes

of the “bridge” protein:

A nucleotide addition cycle:

The bridge is in a straight conformation adjacent to

the nucleotide entry site.

2. After adding a nucleotide to the RNA the bridge

protein is in contacts with the newly added nucleotide,

undergoes a conformational change and moves one base

pair along the template, obscuring the nucleotide entry

site.

3. The bridge returns to its straight conformation, allowing

Entry of next nucleotide of the template - namely,

the bridge acts as a ratchet.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Properties of the core enzyme

The core enzyme of E. coli has a general affinity for DNA (driven

by electrostatic attraction between the basic protein and

the acidic DNA). Yet, it does not distinguish between promoters and

other sequences.

Any random sequence bound by core enzyme is described

as a “loose binding site”. No change occurs in the DNA

which remains duplex.

Such a core enzyme-DNA complex is stable (half life for

dissociation is 60 min.).


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Properties of the holoenzyme

The holoenzyme has a

drastically reduced ability

to recognize “loose binding

sites” (half life of <1sec. Kd

reduced by a factor of 104).

The holoenzyme binds

promoters with Kds 1,000

time higher than core

enzyme with half lives of

hours.

However, it manifests a

specific Kd to any specific

promoter.

Sigma confers the ability to

recognize specific sites. It is

also involved in “melting”,

creating an “open” complex.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Depending of specific promoter the Kd for DNA:RNA pol

association is 106 - 1012 (first level of regulation of rate of

transcription).

Formation of an open complex by melting (that is driven by

sigma) allows tight binding that is not reversible.

Initiation rate (frequency of initiation) also differs

(dependent on other factors in addition to RNA pol:DNA

associatio. Frequencies can range between 1/sec (rRNA genes

to 1/30 min. (lacI promoter).


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

The holoenzyme binds

promoters with Kds 1,000

time higher than core

enzyme with half lives of

hours.

This property assists with promoter

recognition, but significantly interferes

with elongation. Therefore, sigma dissociates

from the enzyme when elongation starts.

Sigma factor is recycled.

It becomes unnecessary when

abortive initiation is concluded.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

(sigma)

(promoter

region)

Sigma contacts mainly bases of the coding strand and continues to

hold these contacts - an important step in melting (forming an “open

complex and recognition of template strand.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

What is responsible for the ability of holoenzyme to bind

specifically to promoters?

Sigma has domains that recognize promoter DNA, but as an independent protein

Sigma does not bind to DNA. There is major change in conformation of sigma

when it binds core enzyme. The N-terminal region of free sigma suppresses the

activity of the DNA-binding region - it is an autoinhibitory domain.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

How holoenzyme finds a specific promoter (60bp in a 4x106 stretch)?

The forward rate constant for RNA Pol binding to promoters is faster than

random diffusion (that limits the constant to 108/M-1Sec-1).

The measured rate constant for association with a 60 bp target

is 1014/M-1Sec-1.

If the target is the whole genome the rate constant is around 1014/M-1Sec-1.

But how does the polymerase move from random binding sites to promoters?

Perhaps RNA Pol binds DNA and remains contact

(no simple diffusion that relies on random binding). Rather, a direct

Displacement with other sequence occurs (no sliding).


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

The “diffusion model: random association with loose sites on DNA,

dissociation and re-bind, until occasionally (statistically) interacting

with a promoter, and remains associated.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

A direct displacement

model - diffusion is not

required


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Promoter’s function is provided directly by

its DNA sequence/structure (it does not need to be

transcribed or translated).

It is a cis-acting site.

[in genetic terminology, sites that are located on the

same DNA are said to be in cis. Sites that are located

on two different molecules of DNA are being in

trans.]


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Conserved - a base most often present at a position.

Perhaps the most striking feature of E. coli promoters is the lack of extensive conservation

of sequence over the 60 bp associated with RNA Pol.

Promoter elements (in E. coli):

Start point (a purine in 90% of the RNAs).

-10 sequence

-35 sequence

The distance separating the -35 and the -10 sites.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

The -10 sequence:

T80A95T45A60A50T96

Sequence that resides in poistions of -18 to -9 in all known

E. coli promoters.

Subscripts denote the percent occurrence of the most frequent found

base


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

The -35 sequence:

T82T84G78A65C54A45

The distance separating the -35 and -10 sites is between 16-18 bp

in 90% of promoters. In the exceptions it can go down to 15 or up

to 20. Sequence itself is not important.

Some promoters have an A-T-rich sequence located farther upstream.

It is called UP element and interacts with a subunit of RNA pol. It

Is typically found in promoters that are highly expressed, such as the

promoters of the rRNA genes.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Up elements are associated with a subunit of RNA pol. Found in promoters that

are highly expressed.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

In spite of conservation of promoters there is ~1000 fold variation

in the rate at which RNA polymerase binds to different promoters

in vitro.

Binding rates correlate well with the frequencies of transcription

in vivo.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Sequences at prokaryotic terminators

show no similarities.

Many terminators require a hairpin to

form.

Termination involves recognition of

signals on the transcript.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Intrinsic terminator - other factors

are not required. Works in vitro

too.

Hairpins may cause polymerase

to slow or even to stop.

Antitermination process may

allow RNA Pol to continue

(readthrough).

Downstream U-rich destabilizes

RNA-DNA hybrid.

Hairpin structure

+ U rich sequence

(1100 sequences in

E. Coli fit these criteria.

Hairpin + U-rich are

Necessary, but not sufficient.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

The weakest base-pair is the rU-dA


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Rho:

A 275 Kd homo-hexamer.

RNA binding domain + ATPase domain.

Belong to a family of ATP-dependent helicases.

Functions as an ancillary factor for RNA Pol.

Most efficient at 10% concentration.

Accounts for about 50% of terminations in E. coli.

Rho-dependent termination sequences are rich in

Cs and poor in Gs. Reside 50-90 bases from

termination sites.

Acts processively on a single RNA substrate.

Moves faster than RNA Pol.

Pausing is important for Rho-dependent termination

too.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Translation

Components involved in translation account for 35% of the dry weight of E. coli cells.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

A condensation reaction: formation of the peptide bond by removal of water (dehydration) from the -carboxyl group of one amino acid and the -amino group of another

-----------------------------------------------------------------------------------


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

To make the reaction thermodynamically more

favorable, the carboxyl group must be

chemically modified or activated so that the

hydroxyl group can be more readily eliminated


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

(Dihydrouridine)


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

First stage in translation: aminoacyl-tRNA synthetases esterify

the 20 amino acids to their corresponding tRNA.

Each enzyme is specific for one amino acid and one or more tRNAs.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Step 1: An enzyme-bound intermediate

Aminoacyl-AMP forms when the carboxyl

group of the amino acid reacts with the

-phosphoryl group of ATP, creating an

anhydride linkage, with displacement of

pyrphosphate.

Step 2: The aminoacyl group is

transferred to its corresponding tRNA.

The resulting ester linkage has a highly

negative standard free energy of

hydrolysis.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Valine and isoleucine differ in only a single methylene group


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

  • Proofreading by aminoacyl-tRNA synthetases

  • Two active sites in the Ile-tRNAIle synthetase:

  • binding of the amino acid to the enzyme (affinity to

  • Ile is only a little higher than affinity to Val (error in

  • 1/200 entries.

  • - binding of aminoacyl-AMP product. This site has higher

  • affinity to AMP-Val. A hydrolytic site.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

What is accomplished by aminoacylation of tRNA?

Activation of the amino acid for peptide bond formation.

2. Attachment of the amino acid to an adaptor tRNA that ensures

appropriate placement of the amino acid in a growing polypeptide.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

N-formyl group is added to the

amino group of methionine by

transformylase.

Transformylase is specific to

Met attached to tRNAfMet


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Translation initiation in prokaryotes

IF-3 prevents combining of the 30S

and 50S subunits

The initiating 5’AUG is guided to its correct

position by the Shine-Delgarno sequence

in the 5’UTR of the mRNA (AUG is the

beginning of an ‘open reading frame’).

The initiating 5’AUG is positioned at a site

called the P site, the only site in the ribosome

to which fMet-tRNAfMet can bind.

The fMet-tRNAfMet is the only aminoacyl-tRNA

that binds first to the P site.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Step 2 in translation initiation:

GTP-bound IF-2 and the fMet-tRNAfMet jointhe ribosome, guided

by the anticodon that pairs with the mRNA initiation codon.

Step 3 in translation initiation:

The complex (30S + IF1,IF2-GTP,IF3 + fMet-tRNAfMet) combines with

the 50S ribosomal subunit; simultaneously, the GTP bound to

IF-2 is hydrolyzed to GDP and Pi which are released from the

complex. All 3 initiation factors are also released from the complex.

IF-2-GDP is re-loaded with GTP via a GDP/GTP exchange reaction.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Translational elongation

Step 1:

Appropriate incoming aminoacyl-tRNA

binds to a complex of GTP bound EF-Tu.

The GTP-EF-Tu-aminacyl-tRNA complex

binds the A site of the 70S complex.

The GTP is hydrolyzed and the EF-Tu-GDP

is released.

EF-Tu-GTP complex is regenerated via a

GDP/GTP exchange reaction catalyzed

by EF-Ts.

AA2


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Translation elongation, Step 2: Formation of the peptide bond:

The -amino group of the amino

acid in the A site acts as a

nucleophile, displacing the tRNA

in the P site to form a peptide bond.

The tRNAfMet at the P site is now

uncharged.

The peptidyl transferase reaction

is probably catalyzed by the

23S rRNA


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Translation elongation, Step 3: Translocation

Move of the ribosome. The ribosome

moves one codon towards the 3’ end of

the mRNA.

Translocation is catalyzed

by EF-G-GTP (translocase).

The ribosome is now ready for the

next elongation cycle.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Termination

Catalyzed by RF1 or RF2.

(depending on the particular stop codon).

RF1 and RF2 are proposed to mimic the

structure of tRNA.

RF-1 recognizes UAG and UAA. RF-2

Recognizes UGA and UAA.

In eukaryotes, a single RF, eRF, recognizes

all 3 termination codons.

Releasing factors:

Hydrolyze the ester linkage of the

peptydil-tRNA bond.

2. Release the polypeptide and the last

tRNA (now uncharged).

3. Dissociate the ribosome to 30S and 50S

subunits.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

EF-Tu EF-G

The carboxy terminal domain of EF-G mimics the structure of tRNA.

Altogether EF-G mimics the structure of EF-Tu-tRNA complex and

probably binds to the A site and displacing the peptidyl-tRNA.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Translation is energy consuming:

On average, hydrolysis of more than 4 NTPs to NDPs is

required for the formation of each peptide bond of a polypeptide.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Bacterial ribosome’s M.W.:

~2.7 million

Components in the ribosome structure:

Proteins: blue (in large subunit); Yellow (in small subunit).

Bases of rRNA in large subunit: white. Backbone of rRNA in large

subunit: green. rRNA in small subunit: white. tRNAs:purpule,

mauve, gray. mRNA contacting tRNAs:red.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

In the 50S subunit, the 5S and 23S rRNAs form the structural core.

The proteins are secondary elements in the complex, decorating the

surface.

No protein is detected within 18A of the active site for peptide

bond formation.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

50S subunit of a bacterial

ribosome.

Red - a puromycine molecule

at the active

peptidyl transferase site. Note

no proteins in the vicinity.


Dudi engelberg room 1 517 tel 658 4718 e mail engelber cc huji ac il

Steady state level of a protein (expression level) is determined

by a combination of regulation of:

Transcription initiation

mRNA degradation (mRNA stability)

mRNA processing

Transport to cytoplasm

Translational control

Folding and protein processing

Protein degradation (protein stability)


  • Login