Cell and molec Chapters 7 (everything RNA)

trying not to die in biol 302

Central Dogma of Molecular Biology

The flow of genetic information in cells from DNA to RNA to protein discovered by Francis Crick

DNA -> RNA

transcription

RNA -> protein

translation

Different gene expression efficiencies

Gene A is transcribed and translated much more efficiently than gene B. This allows the amount of protein A in the cell to be much higher than that of protein B

RNA differs from DNA in that...

It is single-stranded and can fold into a variety of structures. Uracil replaces Thymine. U-A instead of T-A

What bonds do RNA and DNA have in the backbone?

phosphodiester bonds, which form the sugar-phosphate backbone of RNA and DNA

What direction are RNA and DNA read/transcribed?

3' - 5' end

What direction are RNA and DNA created/synthesized?

5' - 3' end

The sugar used in DNA

deoxyribose

The sugar used in RNA

ribose

Difference between Uracil and Thymine

absence of a -CH3 (methyl) in Uracil

How does the chemical linkage between nucleotides differ between RNA and DNA?

The chemical linkage between nucleotides is the same

How can RNA become double stranded?

It often contains short stretches of nucleotides that can basepair with complementary sequences found elsewhere on the same molecule

"Coding" strand of DNA

Complementary to the Template strand. The coding strand is equivalent to the RNA product, minus Uracil. The "top" strand

"Template" strand of DNA

Used as a template for RNA transcription, complementary to the Complementary strand. The "bottom" strand

"Transcript" strand of RNA

The RNA chain produced by transcription. It has the same nucleotide sequence as the Complementary DNA strand

What is RNA polymerase?

An enzyme that transcribes DNA.

What does RNA polymerase do to DNA?

It unwinds the DNA and slides along it.

How are nucleotides added during transcription?

Nucleotides are added one-by-one at the polymerization site in RNA polymerase

How does a RNA/DNA hybrid helix form?

Transiently (briefly) in RNA polymerase during synthesis. "R loop"

Where does RNA transcription occur in Eukaryotes?

Transcription is in the nucleus, then the RNA moves into the cytoplasm to be translated through Nuclear Pores

Where does RNA transcription occur in Prokaryotes?

They don't have a nucleus, so anywhere. They can transcribe and translate at the same time

mRNA (messenger RNA)

code for proteins

miRNA (micro RNA)

regulate gene expression

rRNA (ribosomal RNA)

form the core of the ribosome’s structure and catalyze protein synthesis

tRNA (transfer RNA)

Serve as adaptors between mRNAs and amino acids during protein synthesis. Cloverleaf structure

siRNAs (small interfering RNAs)

Provide protection from viruses. Single stranded, binds with RISC protein and binds to foreign RNA to destroy it. The foreign RNA is cleaved by nuclease Dicer

lncRNA (long noncoding RNA)

“Endogenous noncoding RNAs.” Act as scaffolding in the RNA and may help regulate gene activity

Sigma σ factor

Subunit in Bacterial RNA polymerase, recognizes promoter sites in DNA for transcription. RELEASED when transcription starts. ONLY in Bacteria, and the only subunit needed

Bacteria Promoter site

Where RNA transcription begins. Has more Adenine and Thymine, easier to open. PRINOW BOX

Eukaryote Promoter Site

Where RNA transcription begins. Has more Adenine and Thymine, easier to open. TATA BOX

Terminator site / Stop site

Where RNA transcription stops. Has more Guanine and Cytosine

When does Bacterial RNA poly lose & gain its Sigma Factor?

Releases it after transcription starts, gains it after stopping and looks for another Promoter site

The first nucleotide transcribed in Bacteria is designated as…

+1 (transcription start site)

The direction is transcription is determined by…

The orientation of the promoter at the beginning of each gene

Genes transcribed left to right use the:

bottom DNA strand as a template

Genes transcribed right to left use the:

top DNA strand as a template

How many types of RNA poly. do Bacteria & Eukaryotes have?

Bacteria - one type
Eukaryotes- three types

To initiate transcription, Bacterial RNA polymerase needs..

The sigma subunit, but can initiate on its own

To initiate transcription, Eukaryote RNA polymerase 1,2,3 need..

A large set of accessory proteins around the RNA poly. And general transcription factors that assemble around each promoter

RNA polymerase 1 transcribes:

most rRNA (ribosomal RNA)

RNA polymerase 2 transcribes:

ALL protein-coding genes, miRNA (micro RNAs), lncRNA (noncoding RNA)

RNA polymerase 3 transcribes:

tRNA genes (transfer RNA), 5S RNA, genes for many other small RNA

TF2D

General transcription factor. Recognizes the binds to the TATA Box through one of its subunits called TATA binding protein (TBP)

TF2B

Binds adjacent to TF2D after it binds to the TATA Box

TF2H

pries apart the double helix of DNA at the transcription start point, using the energy of ATP hydrolysis. AND phosphorylates the long polypeptide ”tail” of RNA polymerase II, releasing it from the
general transcription factors so it can begin the
elongation phase of transcription.

Transcription Initiation Complex

Eukaryote RNA polymerase + general transcription factors after assembling at the Promoter

Elongation factors

Help RNA Polymerase 2 move through DNA by prying the Histone off of the DNA. Help RNA Poly 2 transcribe through a Nucleosome

TBP

Subunit of general transcription factor TF2D. Its responsible for finding the TATA Box. Uniquely bends the DNA for transcription which attracts other general transcription factors

β-globin gene

encoding one of the subunits of the oxygen-carrying
protein hemoglobin, contains 3 exons

Factor VIII (8) gene

which encode for a protein (Factor OO) that functions
in the blood-clotting pathway, contains 26 exons. Mutations in this large gene are responsible for the most prevalent form of hemophilia

snRNPs

Small nuclear ribonucleoprotein particles. Part of the Spliceosome. Recognizes the nucleotide sequences at the beginning and end of an Intron. snRNPs contain snRNAs & proteins. FIVE MAIN snRNPs + 200 additional proteins

Phosphorylation of RNA polymerase 2

Done by general transcription factor TF2H. Allows RNA processing proteins to assemble on the tail

RNA Processing

Capping at the 5’ ends with a guanine (5’-5’ seven methyl guanine), polyadenylation at the 3’ends with poly-A tail, and splicing. “Matures” the pre-RNA

RNA Processing and Transcription happen…

Simultaneously in Eukaryotes

Capping

Part of RNA processing. The pre-RNA has a 7-methyl guanine bonded to it’s 5’ end. The guanine is bonded 5’-5’ with a triphosphate bridge

Polyadenation

Cleavage of pre-RNA’s primary transcript at the 3’ end through an enzyme. A second enzyme adds a series of repeating Adenine to the cut end, making a Poly-A Tail

After the 5’ Cap and before the 3’ Poly-A tail, there is a…

Non coding sequence (5’ UTR) and (3’ UTR)

UTR

Untranslated region

RNA transcription and Synthesis mainly happen during…

Interphase. The DNA is not as condensed

Bacterial genes are organized…

a single stretch of uninterrupted nucleotide sequences that encodes the amino acid sequence of a protein

Eukaryotic genes are organized…

the coding sequences of most eukaryotic genes (exons) are
interrupted by noncoding sequences (introns)

Exons

Coding sequences of eukaryotic genes

Introns

Noncoding sequences of eukaryotic genes

Spliceosome

mediates the cleavage of the RNA at the intron-exon borders
and the covalent link of the exons. Takes out the Introns of pre-RNA to “mature” it

What happens during intron splicing? First step

The branch point adenine (A) attacks the 5’ splice site, cutting the RNA backbone. Intron forms a Branched Structure

What happens during intron splicing? Second step

The 5’ end of the intron forms a branched structure by linking to the 2’-OH of the adenine

What happens during intron splicing? Third step

The exons join, and the intron is released as a lariat (cowboy loop), eventually degrading.

Lariat

A cowboy loop formed of introns cut out of pre-RNA by Spliceosome. It’s eventually degraded

U1

snRNP, recognizes the 5′ splice site

U2

snRNP, recognizes the lariat branch-point site

U6

“double-checks” the 5′ splice site by
displacing U1 and base-pairing with the same
intron sequence allowing for a “rereading” step

Alternative RNA Splicing

Different combinations of Exons are used for the mature RNA (mRNA). Allows for various protein production from the same gene

α-tropomyosin

a protein that regulates contraction in muscle cells. Its transcript
can be spliced in different ways to produce distinct mRNAs for different muscles

RNA-binding proteins

signal that a mRNA has been matured and can be moved to the cytoplasm

Exon junction complex (EJC)

Deposited onto mRNA after successful splicing of introns has occurred

Nuclear transport receptor

Moves mRNA from the nucleus to the cytoplasm

Codon

Group of 3 consecutive nucleotides on mRNA, specifies an animo acid. Always written 5’-terminal nucleotide to the left

Wobble effect

An amino acid will have multiple Codons that make it. The first and second nucleotide of the Codons tend to be the same (ex: GAA, GAC)

Initiation / Methionine Codon

AUG

Stop Codons

UAA, UAG, UGA

Reading Frame

How the sequence of nucleotides on mRNA is split up into codons

Direction mRNA is read for translation

5’ - 3’ in sequential sets of three nucleotides

Anticodon

Part of tRNA that has complementary base pairs to the mRNA Codon. Second step of adapting genetic code

Aminoacyl-tRNA synthetase

A protein that couples an animo acid with its corresponding tRNA, they’re linked with ATP. First step of adapting genetic code

Charged tRNA

tRNA coupled with its animo acid within aminoacyl-tRNA synthetase

Two Adaptors that translate the genetic code (mRNA)

First :Aminoacyl-tRNA synthetase
Second: tRNA with its anticodon base-paired to mRNA’s codon

Ribosomes

Found in the cytoplasm of eukaryotes. Has Large subunit & Small subunit. Made of 4 RNAS + 80 proteins. HALF of it’s mass are rRNAs.

How many ribosomal binding sites are for mRNA?

one, in the Small subunit

How many ribosomal binding sites are for tRNA?

three. A, P, E sites, in the Large subunit.
aminoacyl-tRNA, peptidyl-tRNA, and exit,

How many steps is a Translation cycle?

Four.

Step one of Translation in Ribosome

tRNA binds: tRNA with the next amino acid binds to the A-site on the ribosome.

Step two of Translation in Ribosome

Peptide bond formation: The polypeptide chain is linked to the new amino acid at the A-site by a peptide bond, catalyzed by 23S rRNA (a ribozyme).

Step three of Translation in Ribosome

Subunit shift: The large ribosomal subunit shifts, moving tRNAs to the E- and P-sites.

Step four of Translation in Ribosome

Ribosome resets: The small subunit moves along the mRNA, resetting the ribosome for the next tRNA to bind at the A-site.

Translation Termination in Eukaryotes

Signaled by a release factor (stop codon) on the end of mRNA entering the A-site of the Ribosome

Release Factor

Bind to stop codon and alters peptidyl transferase, catalyzing the addition of water. The reaction frees the carboxyl end of the polypeptide chain being made by the tRNA, releasing it

Translation Initiation in Eukaryotes

Initiator tRNA coupled to methionine (via Start Codon), loaded into Small subunit P-site. It keeps shifting along mRNA until it finds AUG, then initiation factors dissasociate, the Large subunit attaches. Synthesis begins when the enxt charged tRNA enters the A-site

Operons

In prokaryotes, a cluster of genes that are transcribed together into one mRNA. Several proteins can be synthesized from this one mRNA

Eukaryotic ribosomes recognize a…

5’ cap