Chapter 10 (DNA Transcription and Translation)

Three steps of transcription

1. Initiation
2. Elongation
3. Termination

Initiation

Beginning of transcription. It occurs when the enzyme RNA polymerase binds to a region of a gene called the promoter. This signals the DNA to unwind so the enzyme can ''read'' the base

Elongation

Addition of nucleotides to the mRNA strand. DNA polymerase reads the unwound DNA strand and builds the mRNA molecule, using complementary base pairs

Termination

Ending of transcription, and occurs when RNA polymerase crosses a stop (termination) sequence in the gene. The mRNA strand is complete, and it detaches from DNA.

Eukaryotes have three polymerases that are each made up of ________ subunits or more

10

RNA polymerase I (Insensitive)

located in the nucleolus and synthesizes all rRNA EXCEPT for the 5S rRNA molecule.

The rRNA molecules are considered ___________ RNAs because they have a cellular role but are not translated into protein.

Structural RNAs

RNA polymerase II (extremely sensitive)

Located in the nucleus and synthesizes all protein-coding nuclear pre-mRNA

RNA polymerase III (Moderately sensitive)

Located in the nucleus. This polymerase transcribes a variety of structural RNAs that includes the 5S pre-rRNA, transfer pre-RNAs (pre-tRNAs), and small nuclear pre-RNAs.

tRNAs have a critical role in translation:

They serve as the adaptor molecules between the mRNA template and the growing polypeptide chain

Small nuclear RNAs have a variety of functions, including

"Splicing" pre-mRNAs and regulating transcription factors.

Eukaryotic and prokaryotic promoters both have a

TATA box

____________ transcription factors are crucial in the formation of a preinitiation complex on the DNA template that subsequently recruits RNA polymerase II for transcription initiation.

Basal

(Elongation process) These DNA-histone complexes, collectively called nucleosomes, are regularly spaced and include _________ nucleotides of DNA wound around eight histones like thread around a spool.

146

FACT

Facilitates chromatin transcription

What does FACT do?

Pulls histones away from the DNA template as the polymerase moves along it. Once the pre-mRNA is synthesized, the FACT complex replaces the histones to recreate the nucleosomes.

The three most important steps of pre-mRNA processing are:

The addition of stabilizing and signaling factors at the 5′ and 3′ ends of the molecule, and the removal of intervening sequences that do not specify the appropriate amino acids.

5' Capping

the addition of 7-methyl guanosine to the 5' end

3' Poly A-tail

In eukaryotes, a series of 1-200 adenine residues added to the 3' end of an mRNA; the tail appears to enhance the stability of the mRNA by protecting it from degradation.

Exons

Correspond to protein-coding sequences (ex-on signifies that they are expressed), and intervening sequences

Introns

May be involved in gene regulation but are removed from the pre-mRNA during processing. Intron sequences in mRNA do not encode functional proteins.

For the most part, the sequences of __________ can be mutated without ultimately affecting the protein product.

Introns

Splicing

Process of removing introns and reconnecting exons

What does splicing ensure?

Introns will be removed and exons rejoined with the accuracy and precision of a single nucleotide. The splicing of pre-mRNAs is conducted by complexes of proteins and RNA molecules called spliceosomes.

Alternative splicing

This regulated process makes different mRNAs and proteins from the same initial RNA transcript.

miRNA

Can cause mRNA degradation or block translation. Directs the protein complex to "matching" mRNA molecules (ones that form base pairs with the miRNA)

When a eukaryotic gene is transcribed in the nucleus, the primary transcript (freshly made RNA molecule) isn't yet considered a messenger RNA.

pre-mRNA

When the RNA-protein complex binds:

If the miRNA and its target match perfectly, an enzyme in the RNA-protein complex will typically chop the mRNA in half, leading to its breakdown.
If the miRNA and its target have some mismatches, the RNA-protein complex may instead bind to the mRNA and keep it from being translated.

What is the first mRNA processing step?

5' capping

Different mRNAs are made from the same initial pre-mRNA by ________.

Alternative splicing

________ protect(s) the pre-mRNA molecule.

3' Poly A tail

Initiator tRNA

Interacts with the start codon AUG, links to a formylated methionine called fMet, and can also bind IF-2

In E. coli mRNA, a sequence upstream of the first AUG codon, called the ________________________________

Shine-Dalgarno sequence (AGGAGG)

Shine-Dalgarno sequence (AGGAGG)

Interacts with the rRNA molecules that compose the ribosome. This interaction anchors the 30S ribosomal subunit at the correct location on the mRNA template.

GTP

Guanosine triphosphate, which is a purine nucleotide triphosphate, acts as an energy source during translation—both at the start of elongation and during the ribosome's translocation.

Kozak's rules

The nucleotides around the AUG indicate whether it is the correct start codon.Essentially, the closer the sequence is to this consensus, the higher the efficiency of translation.

The formation of each peptide bond is catalyzed by _______________________

Peptidyl transferase

Peptidyl transferase

An RNA-based enzyme that is integrated into the 50S ribosomal subunit

Termination of translation occurs when a nonsense ___________ is encountered

Codon

Nonsense codonswhen termination of translation

UAA, UAG, or UGA

The process of translation, or protein synthesis, involves the decoding of an mRNA message into a _____________ product.

Polypeptide

Polysome.

The complete mRNA/poly-ribosome structure

The tRNAs are structural RNA molecules that were transcribed from genes by which RNA polymerase?

RNA polymerase III

How many tRNAs exist in the cytoplasm

40 to 60 types of tRNAs exist in the cytoplasm

Of the 64 possible mRNA codons—or triplet combinations of A, U, G, and C—three specify the termination of protein synthesis and 61 specify the addition of amino acids to the _____________________

Polypeptide chain

Encodes the initiation of translation.

AUG

tRNAs need to interact with three factors

They must be recognized by the correct aminoacyl synthetase.
They must be recognized by ribosomes.
They must bind to the correct sequence in mRNA.

How many amino acids?

20

Aminoacyl tRNA synthetase

The process of tRNA "charging," each tRNA molecule is linked to its correct amino acid by a group of enzymes

Aminoacyl tRNA synthetase function

Enzymes first bind and hydrolyze ATP to catalyze a high-energy bond between an amino acid and AMP

AMP

Adenosine monophosphate

AMp function

A pyrophosphate molecule is expelled in this reaction. The activated amino acid is then transferred to the tRNA, and AMP is released.

Some of the bases of pre-___________ are methylated and Pre-tRNA molecules also undergo methylation

rRNAs

Mature rRNAs make up approximately _____ % of each ribosome

50

Mature tRNAs take on a three-dimensional structure through intramolecular hydrogen bonding to position the amino acid binding site at one end and the ______________ at the other end

Anticodon

Anticodon

A three-nucleotide sequence in a tRNA that interacts with an mRNA codon through complementary base pairing.

Nucleotide doublets would not be sufficient to specify every amino acid because there are only 16 possible two-nucleotide combinations (42). In contrast, there are ______possible nucleotide triplets (43), which is far more than the number of amino acids.

64

Degenerate

A given amino acid could be encoded by more than one nucleotide triplet

Codons

Nucleotide triplets

_______________ is believed to be a cellular mechanism to reduce the negative impact of random mutations

Degeneracy

Signal sequence

A short tail of amino acids that directs a protein to a specific cellular compartment

Proteins can be chemically modified with the addition of groups including

Methyl, phosphate, acetyl, and ubiquitin groups

Proteasome

An organelle that functions to remove proteins, to be degraded

One way to control gene expression, therefore, is to alter the __________ of the protein.

Longevity

Proteins are moved to their final destination in a cell by way of ________.

Signal sequences

tRNAs begin as ________ molecules that are transcribed and processed in the ________.

pre-tRNA, nucleus

Chaperones help proteins ________

fold properly

A given amino acid could be encoded by more than one nucleotide triplet. This indicates that the genetic code is _______________

Degenerate

Ribosomes contain catalytic ________ molecules.

rRNA

There are ________ stop (nonsense) codons.

Three

When the A site encounters a ________ codon, translation end

Nonsense

Translation always begins by adding a(n) ________ to the beginning of every polypeptide chain.

Methionine

tRNAs are normally ________ when not involved in active protein synthesis.

free in the cytoplasm

tRNAs contain the anticodon that interacts with ____________

mRNA.

Plasmids (Prokaryotes)

Shorter circular DNA molecules that may only contain one or a few gene.Can be transferred independently of the bacterial chromosome during cell division and often carry traits such as antibiotic resistance

Prokaryotic RNA Polymerase

The polymerase is composed of five polypeptide subunits, two of which are identical. Four of these subunits, denoted α, α, β, and β′ comprise the polymerase core enzyme

Holoenzyme.

The polymerase comprised of all five subunits

Promoter

A DNA sequence onto which the transcription machinery binds and initiates transcription

Promoter consensus sequences

Regions that are similar across all promoters and across various bacterial species

Elongation and Termination in Prokaryotes

The RNA polymerase acts as a stable linker between the DNA template and the nascent RNA strands to ensure that elongation is not interrupted prematurely.

Rho-dependent termination

Controlled by the rho protein, which tracks along behind the polymerase on the growing mRNA chain

Rho-independent termination

Controlled by specific sequences in the DNA template strand

Hairpin

Causes the polymerase to stall as soon as it begins to transcribe a region rich in A-T nucleotides. The complementary U-A region of the mRNA transcript forms only a weak interaction with the template DNA

Initiation of protein synthesis (Prokaryotes)

Begins with the formation of an initiation complex. Three initiation factors that help the ribosome assemble correctly, guanosine triphosphate (GTP) that acts as an energy source

Elongation of translation (Prokaryotes)

- tRNA-AA loaded into A site of the ribosome
- if new tRNA in A site is a match, accepted --> conformational change of A site
- then GTP on EF-Tu is hydrolyzed, and EF-Tu is released and recycled
- AA end of tRNAs in P and A sites in close proximity:
- peptide bond formed
- f-Met transferred to AA on tRNA in A site (peptide bond), EF-G appears, fits into A site, shifts tRNA into new site
- ribosome moves forward

A (aminoacyl) site

Binds incoming charged aminoacyl tRNAs

P (peptidyl) site

Binds charged tRNAs carrying amino acids that have formed peptide bonds with the growing polypeptide chain but have not yet dissociated from their corresponding tRNA

E (exit) site

Releases dissociated tRNAs so that they can be recharged with free amino acids

Peptidyl transferase

An RNA-based ribozyme that is integrated into the 50S ribosomal subunit

Promoters in prokaryotes consist of ________

Two consensus sequences

In prokaryotes, the ________ anchors the 30S ribosomal subunit to the correct location on the mRNA template.

Shine-Dalgarno sequence

Elongation of the transcript in prokaryotes requires the ________.

Core-enzyme

Central Dogma of Life

DNA dictates the structure of mRNA in a process known as transcription, and RNA dictates the structure of protein in a process known as translation

More information on Central Dogma of Life

DNA is transcribed to RNA via complementary base pairing rules (but with U instead of T in the transcript)
The RNA transcript, specifically mRNA, is then translated to an amino acid polypeptide
Final folding and modifications of the polypeptide lead to functional proteins that actually do things in cells

_______ serve(s) as evidence that life may have evolved before the central dogma.

Catalytic RNA

According to the central dogma, ________.

DNA is transcribed to RNA