Lecture 42 learning goals

Understand the following key points

Notes on gene orientation

• In double-stranded DNA, the top strand is written in ___, while the bottom strand is written in __ direction.

A gene starts at a point where ___ starts and is called 5'-end.

Moving along a gene in the 5' - 3' is called ____ while moving in the 3' - 5' is called ____.

Transcription start site of a gene in double-stranded DNA is written on
the __, while the termination site is written on the ___.

• In double-stranded DNA that codes for a gene, the top stand is called the ___ strand (___ strand) since its sequence corresponds to the transcribed RNA product.

On the other hand, the bottom strand is
called ___ strand (____ strand) since it serves as a ___ for gene transcription.

The enzymes that catalyze RNA synthesis are called RNA _____

E. coli RNA polymerase is composed
of ___ core polypeptides with a
stoichiometry of α2ββ'ω.

All ___ eukaryotic polymerases have
five core polypeptides homologous to
the E. coli RNA polymerase.

• Also, all eukaryotic RNA pols share
___ common subunits, and each
contains three to seven unique smaller
subunits.

Gene transcription by RNA pols

Unlike DNA polymerases, RNA polymerases can initiate RNA synthesis by ___ in the presence of initiation factors such as subunits in prokaryotes and the general transcription factors in eukaryotes.

• ____: DNA sequences in genes that often lie upstream of transcription start site and recruit RNA pols for transcription.

Promoter consensus sequences for different genes ___.

• Genes with __-rich consensus sequences are frequently transcribed (e.g. rRNA operon) and are considered strong promoters, while those with __-rich are less frequently transcribed (aka weak promoters).

Transcription initiation

RNA pol binds to the ___, unwinds DNA, induces transcription bubble formation, positions template at the active site, and starts transcription.

NTPs that diffuse to the active site base pair with the template at the +1
and +2 position, leading to the formation of a 3'-5' ___ bond.

RNA synthesis is almost
identical to DNA synthesis

Reaction catalyzed by RNA Pol.
___, not dNTPs are used

• Incoming NTP base pairs

• Nucleophilic attack by the _-__ group on
the α-phosphate of the NTP

• 3'-5' ____ bond formation

• Release of the βγ phosphate from the NTP as pyrophosphate (PPi).

• Hydrolysis of the PPi by ____, providing an additional thermodynamic driving force that favors the reaction.

• In E. coli, the rate of transcription is 30-85 NTPs per second, which is ___ than DNA synthesis.

Elongation of RNA synthesis

Following the formation of the first phosphodiester bond, RNA polymerase
"walks" along one strand of DNA, known as the ___ strand, in the
3' to 5' direction.

For each nucleotide in the template, RNA polymerase adds a matching (complementary) RNA nucleotide to the _ end of the RNA strand.

After adding about 10 nucleotides, RNA pol holoenzymes move away from the promoter region. This step is known as promoter ___.

Synthesis continues until the polymerase encounters a ____ signal.

Termination of RNA synthesis

There are two major types in bacteria: __-___ and ___-___

In _-___ termination, the RNA contains a binding site for a protein called Rho factor.

Rho factor binds to this sequence, travels
towards RNA polymerase, and induces ____, leading to the release of RNA.

The Rho factor is helped by the presence of a __-rich transcription-stop point in the DNA that pauses the RNA pol.

Rho-independent termination:

Close to the termination site, RNA has
complementary GC-rich two-fold symmetry sequence that folds back on itself and forms a stable ___ that causes the polymerase to ___.

The hairpin is followed by a series of _ nucleotides in the RNA, which makes weak base pairing with As in the DNA template, allowing ___ of the RNA transcript.

Coupled with the ___ polymerase, weak pairing causes ___ and
release of the new RNA transcript

Termination of transcription in EUKARYOTES

• More complex and not fully understood

• In case of mRNA transcription, the process involves appearance of __-_ signal sequence in the RNA transcript

• Steps in mRNA termination: binding of CPSF (cleavage and poly-A signal factor) to ___ signal sequence, cleavage of the nascent transcript, and addition of __-__ ___

RNA particularly tRNA, rRNA, and mRNA
are ____ before use in cells

Post-transcriptional tRNA processing

(a) RNase P and other ____
cleave the primary transcript

Post-transcriptional tRNA processing

(b) RNase __ trims the 3' end.

Post-transcriptional tRNA processing

(c) tRNA nucleotidyl transferase adds
___ to the 3' end.

rRNA processing

In prokaryotes, rRNA is produced as a single transcript and then cleaved
by the actins of __ __ ____ to mature 16S, 23S, and 5S.

Endonucleases M16, M23, and M5 trim the __ ___ to final mature forms.

• In eukaryotes also, three of the rRNAs are produced by cleavage of a ___ transcript — 18S, 5.8S and 28S are produced.

The fourth rRNA in eukaryotes, 5S, is produced as a monomer by __ ___ __ and is processed separately

Note: ___ unit (symbol S, sometimes Sv) is a non-metric unit for the sedimentation coefficient. The ___ unit (S) offers a measure of a particle's size based on its sedimentation rate, i.e. how fast a particle of given size and shape '___' to the bottom of a solution.

Eukaryotic rRNA processing

The 45S pre-rRNA is transcribed in the ___ and processed into the
mature 18S, 5.8S and 28S rRNAs by a series of ____ and ____ steps.

Note that methylation of regions that become mature rRNA occurs BEFORE removing the ___.

Messenger RNA processing

• In prokaryotes, translation begins even before transcription is ___.

• In eukaryotes transcription occurs in the ___ and translation occurs in the ___.

____ allows eukaryotes to process mRNA.

• In eukaryotes, primary mRNA transcripts are processed in the following ways:

1. 5'-end ____
2. 3'-end ____
3. Splicing ___ and removing ___

Capping of the 5'-end of mRNA
Formation of a cap at the 5' end of mRNA.

The 5' end of eukaryotic mRNA
is a nucleoside triphosphate initiated by RNA pol __. This end is capped to increase ___ and suitability for translation.

Steps

• Removal of the γ-phosphate by ____.

Capping with GTP by ___ ___ to create a 5'-5' GTP linkage

• ____ of the G at N-7 and a few successive 2' oxygen atoms of ribose.

Polyadenylation

Steps
The appearance of __-__ signal

Recognition of poly-A signal by ___ (Cleavage and Poly-A Signal Factor)

Polyadenylation by poly-A ___ and release

Note: Polyadenylation enhances mRNA ___ and transport to the cytoplasm
where translation takes place.

Splicing __ to produce mature mRNA

General scheme of splicing:

In eukaryotes, primary mRNA
transcripts are much larger, and mature ones are produced by what is known as
splicing of ___ and removal of ___.

mRNA Splicing

The splicing of two successive exons in a nascent mRNA transcript requires removal a single ___, using two ____ reactions:

1. between the 5' splice site and the branch site adenylate

AND

2. between the 5' exon and the 3' splice site

The products of these reactions are ___ exons and an ___ intron

These reactions are catalyzed by a large RNA-protein complex called the
___ composed of over 100 proteins and 5 RNA molecules.

The RNA molecules are called ___ __ ___ (__) and when they associate with proteins they are called small nuclear ribonucleoproteins (snRNAPs) and is sometimes referred to as ___.

The five snRNAs are named U1, U2, U4, U5, and U6 (U stands for ___ which is common in the snRNAs).

(a) The spliceosome positions the adenylate residue at the branch site near the __ splice site. The 2'-hydroxyl group of the adenylate attacks the __ splice site.

(b) The 2'-hydroxyl group is attached to the 5' end of the ___, and the newly created 3'-hydroxyl group of the exon attacks the __ splice site.

(c) As a result, the ends of the exons are joined, and the intron, a ___-shaped molecule, is ___.