DNA, RNA, Proteins (Transcription and Translation)

DNA template-directed duplication of the genome prior to cell division

DNA replication

DNA template-directed biosynthesis of RNA

Transcription

RNA template-directed biosynthesis of DNA

Reverse transcription

mRNA template-directed biosynthesis of proteins

Translation

encode the amino acid sequences of all the polypeptides found in the cell.

Messenger RNAs

match their anticodon to the mRNA while carrying a specific amino acid used for protein synthesis

Transfer RNAs

are constituents of the large and small ribosomal subunits.

Ribosomal RNAs

regulate the expression of genes, possibly via binding to specific nucleotide sequences.

MicroRNAs

_____ are catalytic RNA molecules that act as enzymes

Ribozymes

Processing of mRNAs

____ - elimination of introns; joining of exons

______of the 3’ end

______ the 5’ end

Splicing, polyadenylation, capping

The synthesis (of transcription in E.coli) is catalyzed by enzyme (RNA polymerase) and involves 2 _____ ions for catalysis.

Mg

binds to a sequence called promoter sequence (or just promoter) to begin transcription.

primer not required

forms a bubble

generates positive supercoils ahead, later relieved by topoisomerases.

RNA polymerase (RNA Pol)

RNA Pol has no Proofreading Capability

lacks 3’ → 5’-exonuclease (high error rate)

the _ subunit is the main catalytic subunit and is responsible for DNA binding

the _ subunit directs enzyme to the promoter. Each class of RNA pol holoenzymes has a different  subunit.

The _ appears to protect the polymerase from denaturation.

B, o, w

There are two consensus sequences at −10 (TATAAT) and −35 (TTGACA) for o subunit binding

TATA sequences

An ____−rich upstream promoter element between −40 and −60 binds the a subunit.

______−rich sequences promote strand separation.

These sequences govern efficacy of RNA Pol binding and therefore affect gene-expression level.

A-T

Initiation of Transcription: RNA polymerase first forms a closed-complex in which DNA is not unwound

Next, the stable, open complex forms where the −10 region is unwound

Lastly, an ___________ further unwinds the DNA from −12 to +2

Mg dependent isomerization

Elongation – The Transcription Bubble

• RNA chain growth begins by binding the template-specific rNTP, followed by binding of the second

  Nucleophilic attack by the 3′-OH of the first nucleotide on the α-phosphate of the second nucleotide forms a _____ bond in a manner analogous to DNA replication

• As this process continues, the DNA is continuously unwound in a “transcription bubble”

  σ-factor dissociates during incorporation of the first 10 nucleotides

phosphodiester

Two Types of Termination in E. coli

• p-independent

• characterized by _____ near the 3’ end of the transcript

  self-complementary regions in transcript form a hairpin 15−20 bp before the 3’ end

  makes the RNA Pol pause, dissociate

UUU

Two Types of Termination in E. coli

2. p-dependent

common CA-rich sequence called a _____ (Rho utilization element)

protein processes until termination site reached

protein is a helicase, binds to rut site

rut site

Regulation of Transcription

_____ – deviating from the consensus sequence

promoter sequence

Regulation of Transcription

_____– cAMP receptor protein (CRP)

activator proteins

Regulation of Transcription

_______ – block necessary binding sites

repressor proteins

Transcription in Eukaryotes Utilizes Three Polymerases

• RNA polymerase ? transcription of the major ribosomal RNA genes (precursor for 28S, 18S, and 5.8 rRNAs)

I

Transcription in Eukaryotes Utilizes Three Polymerases

• RNA polymerase ? – transcription of protein-coding genes and some small RNA genes

  very fast (500–1000 nucleotides/sec)

  can recognize thousands of promoters

II

Transcription in Eukaryotes Utilizes Three Polymerases

RNA polymerase ? – transcription of small RNA genes

III

Assembly of RNA Polymerase II at Promoter

• ____ activity in TFIIH unwinds DNA at the promoter.

Helicase

Assembly of RNA Polymerase II at Promoter

• ___ activity in TFIIH phosphorylates the polymerase at the CTD (carboxy-terminal domain), changing the conformation and enabling RNA Pol II to transcribe.

Kinase

Neutralization of the basic _____ residues via acetylation weakens the ionic interactions between histones and DNA in chromatin

lysine (Lys,K)

7-methylguanosine links to 5’-end via 5’,5’-triphosphate link.

formed with a molecule of GTP

may include additional methyl-ations at 2’OH groups of next two nucleotides following the cap

methyl groups derive from S-adenosylmethionine (SAM)

Protects RNA from nucleases

Forms a binding site for ribosome

the 5’-Cap

exons

coding region

introns

non-coding region

Group I and group II introns are _____

require no additional proteins or ATP

in nuclear, mitochondrial, and chloroplast genomes

differ mainly in the splicing mechanism

found in some bacteria

self-splicing

Spliceosomal introns are spliced by enormous complexes called _____.

the most common introns

frequent in protein-coding regions of eukaryotic genomes

spliceosomes

_____ are spliced by protein-based enzymes.

Primary transcript is cleaved by ________.

Exons are joined by ATP-dependent ligase

tRNA introns, endonucleases

Spliceosome Introns

• Spliceosome Introns Are Removed via a Large Complex Called a Spliceosome

• The spliceosome is made up of ______ (“snurps” for small nuclear ribonuclear proteins).

  snRNP RNA is called snRNA (for small nuclear RNA), which are 100−200 nt long.

  five snRNAs known in eukaryotes (U1, U2, U4, U5, U6)

  ______ usually mark sites of splicing.

snRNPs, GU at 5’-end and AG at 3’-end

MicroRNAs (miRNAs):

short noncoding RNAs of ~22 nucleotides

bind to specific regions of mRNA to alter translation

assist in cleaving the mRNAs

or block the mRNA from translation

About 1,500 human genes encode miRNAs and 1 or more affect the expression of MOST protein-coding genes!

Synthesized from larger precursors

processed by two endoribonucleases

__

Drosha and Dicer

The RNA lifetime is one means of gene regulation.

Each gene product is needed for a unique amount of time

Half-lives vary from seconds to hours.

rate of synthesis : rate of degradation

steady state – rates are equal and balanced

typical vertebrate mRNA ~3 hrs

~10 turnovers per cell generation

shorter (~1.5 mins) half-lives for bacterial mRNAs

Degradation occurs via ______.

Hairpin structures in bacterial mRNA can extend half-life.

In eukaryotes, the 5’cap and 3’ poly (A) tail aid in the stability of the mRNA.

ribonucleases

Most DNA and RNA is synthesized from a DNA template; however, viruses do not fit with the norm.

Retroviruses have genomes of ssRNA and the enzyme ______.

virus enters host cell

reverse transcriptase makes DNA from the RNA

It then degrades the RNA from the DNA-RNA hybrid and replaces it with DNA.

DNA can then be incorporated into host DNA.

reverse transcriptase

Retroviruses Typically Contain

• encodes a long polypeptide that is cleaved into six smaller proteins that make up viral core

gag (group associated antigen)

Retroviruses Typically Contain

• encodes protease that cleaves the long polypeptide, reverse transcriptase, and an integrase to insert DNA into host genome

Pol (polypeptide)

Retroviruses Typically Contain

• encodes viral envelope

env

Retroviruses Typically Contain

• _ facilitates integration of virus genome into host DNA

Long terminal repeat (LTR)

Pharmaceutical Targets for HIV (Antiretroviral Drugs)

• ____

  nucleotide or nucleoside analogs

  drug names ending in “dine” or “sine”:

  zidovudine (AZT), didanosine (Videx), and so on

Reverse transcriptase inhibitors, dine/sine

Pharmaceutical Targets for HIV (Antiretroviral Drugs)

• ______

since proteases are used in cleaving proteins for packaging into new viral particles

drug names ending in “avir”:

indinavir, saquinavir, and so on

Protease inhibitors, avir

stop codon

UAA, UAG, UGA

start codon

MET

The third base of a codon can form noncanonical base pairs with its complement (anticodon) in tRNA.

Some tRNAs contain Inosinate (I), which can H-bond with U,C, and A.

These H-bonds are weaker and were named by Crick as “wobble” base pairs.

Example: In yeast, CGA, GCU, and CGC all bind to tRNAArg, which has the anticodon 3’-GCI-5’.

Although sequences are usually written 5’3’, the anticodon here is written 3’5’ to illustrate its bonding to the mRNA codons.

wobble

The alignment of two RNA segments is ____.

antiparallel

Characteristics of tRNAs

• ss RNA of 73–93 nucleotides in both bacteria and eukaryotes

  Cloverleaf (2D), “Twisted L” (3-D)

• _____

Most have G at 5’-end; all have CCA at 3’-end

Characteristics of tRNAs

• has amino acid esterified via carboxyl group to the 2’-OH or 3’-OH of the A of the terminal CCA codon

Amino acid arm

Characteristics of tRNAs

• _____

  D arm

  contains dihydrouridine (D)

  contributes to folding

  TC arm

  contains pseudouridine ()―has bonding between base and ribose

  helps in folding

Anticodon arm helps in folding

Translation Stage 1, Step 1:

• _____esterify 20 amino acids to corresponding tRNAs.

  COO– of amino acid attacks phosphate of ATP  creates aminoacyladenylate intermediate

  Pyrophosphate (PPi) is also cleaved, so the reaction is driven forward by two phosphoanhydride bond cleavages.

  The fate of the aminoacyladenylate varies.

creation of aminoacyl intermediate, Aminoacyl-tRNA synthetases

Translation Stage 1, Step 2:

• _______ (two classes) transfer aminoacyl group from enzyme to tRNA.

  2’-OH or 3’-OH of tRNA attacks phosphate of aminoacyl intermediate, creating phosphodiester bond between amino acid and tRNA

transfer of aminoacyl to tRNA, Aminoacyl-tRNA synthetases

Five Stages of Protein Synthesis

1. ______

   • tRNA aminoacylated

2. ______

   • mRNA and aminoacylated tRNA bind to ribosome

3. _____

   • cycles of aminoacyl-tRNA binding and peptide bond formation…until a STOP codon is reached

4. ______

   • mRNA and protein dissociate, ribosome recycled

5. _____

   • catalyzed by a variety of enzymes

Activation of amino acids, Initiation of translation, Elongation, Termination and ribosome recycling, Folding and posttranslational processing

Each enzyme (specific synthetase) binds a specific amino acid and the matching tRNA.

Aminoacyl-tRNA Synthetases

Translation Stage 2

• The first tRNA is unique.

  The first codon of any peptide is AUG (Met).

  All organisms have two tRNAs for Met.

  Prokaryotic organism (e.g. bacteria) protein starts with fMet-tRNA

  N-formylmethionine (special type)

  Interior Met is inserted with normal tRNA-Met.

  In bacteria, initiation requires

  30S and 50S ribosomal subunits

  mRNA

  fMet-tRNA

  initiation factors IF-1, IF-2, and IF-3

  GTP and Mg+2

  Eukaryotic protein begins with Met, not fMet, but a special tRNA is still used for peptide initiation.

Initiation

Stage 2: Initiation, Steps 1 and 2

• Step 1: _______

  Initiation factor IF-3 keeps 30S and 50S subunits apart.

  The initiating (5’)-AUG codon of mRNA is guided to its correct position by the Shine−Dalgarno sequence

  region in mRNA that is complementary to a sequence in ribosomal RNA.

  Step 2:: _______

  Formylmethionine tRNA binds to the peptidyl (P) site along with initiating(5’)AUG.

The 30S ribosomal subunit binds IF-1, IF-2, and IF-3 and mRNA, fMet-tRNAfMet joins the complex.

Stage 2: Initiation, Step 3

• Step 3: _______

  Large 50S subunit combines with the 30S subunit forming the initiation complex.

  IF-2 hydrolyzes GTP.

  Eukaryotes have a similar process, but the mechanistic details are different and more complex

50S subunit associates

Stage 3: _____ (Prokaryotic)

Step 1: _______

Incoming aminoacyl-tRNA binds first to an EF-Tu –GTP complex.

The aminoacyl-EF-Tu-GTP complex binds to the aminoacyl (A) site of the 70S initiation complex.

After GTP hydrolysis, EF-Tu-GDP leaves the ribosome.

Step 2: ______

There are now two amino acids bound to tRNAs positioned for joining.

One is on the A-site, the other on the P- site.

N-formylmethionyl group is transferred from its tRNA in the P site to the amino acid in the A site.

The reaction is catalyzed by the 23S rRNA (ribozyme).

Ribozyme are self-catalyzing proteins

“Uncharged” (deacetylated) tRNA-fMet is now in the P site.

Step 3: _______

The ribosome moves one codon toward the 3’-end of the mRNA.

uses energy from GTP hydrolysis

GTP is part of EF-G (translocase)

leaves A site open for new aminoacyl-tRNA

Elongation, binding of the incoming aminoacyl-tRNA, peptide bond forms, translocation of the ribosome

Stage 4: _____

Signaled by a stop codon

UAA, UAG, or UGA in the A site will trigger the action of termination factors (release factors) RF-1, RF-2, RF-3.

These help to:

hydrolyze terminal peptide-tRNA bond

release peptide and tRNA from ribosome

cause subunits of ribosome to dissociate so that initiation can begin again

Termination

Antibiotic Drugs

similar structure to 3’-end of aminoacyl-tRNA

binds to the A site of ribosomes, forming bond with growing peptide

Inhibits translocation, so arrests protein synthesis

Puromycin

Antibiotic Drugs

• _____

  They block the A site on the ribosome.

Tetracyclines

Antibiotic Drugs

• _______

  They block peptidyl transfer.

  inhibits mitochondrial, bacterial and chloroplast ribosomes

Chloramphenicol and cycloheximide

Antibiotic Drugs

• It causes code to be misread and inhibits initiation at high concentrations.

Streptomycin

As peptide emerges from the ribosome, the signal sequence is bound by ______

SRP/ribosome/RNA complex is delivered to the ER lumen.

Some modification takes place here (glycosylation, etc.).

Transport vesicles then take proteins to Golgi apparatus.

Proteins are sorted here in ways poorly understood.

Proteins for mitochondria and chloroplast bind chaperone proteins in the cytosol and are delivered to receptors on the exterior of the organelle.

Proteins are eventually degraded to recover the original amino acids

Uses _____

signal recognition particle (SRP), ubiquitine